Cross-reactive t cell epitopes of hiv, siv, and fiv for vaccines in humans and cats

ABSTRACT

The subject invention concerns methods and materials for inducing an immune response in an animal or person against an immunodeficiency virus, such as HIV, SIV, or FIV. In one embodiment, a method of the invention comprises administering one or more antigens and/or immunogens to the person or animal wherein the antigen and/or immunogen comprises one or more evolutionarily conserved epitopes of immunodeficiency viruses. In one embodiment, the epitope is one that is conserved between HIV and SIV, or between HIV and FIV. In another embodiment, the epitope is one that is conserved between HIV, SIV, and FIV.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersR01-AI65276 and R01-AI30904 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of InternationalApplication No. PCT/US2013/054191, filed Aug. 8, 2013, which claims thebenefit of U.S. Provisional Application Ser. No. 61/841,122, filed Jun.28, 2013, U.S. Provisional Application Ser. No. 61/684,592, filed Aug.17, 2012, and U.S. Provisional Application Ser. No. 61/681,014, filedAug. 8, 2012, each of which is hereby incorporated by reference hereinin its entirety, including any figures, tables, nucleic acid sequences,amino acid sequences, or drawings.

The Sequence Listing for this application is labeled 2E99114.txt whichwas created on Jan. 29, 2015 and is 159 KB. The entire contents of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

An effective prophylactic HIV-1 vaccine is needed to eradicate theHIV/AIDS pandemic but designing such a vaccine is a challenge. Despitemany advances in vaccine technology and approaches to generate bothhumoral and cellular immune responses, major phase-II and -III vaccinetrials against HIV/AIDS have resulted in only moderate successes. Themodest achievement of the phase-III RV144 prime-boost trial in Thailandre-emphasized the importance of generating robust humoral and cellularresponses against HIV. While antibody-directed approaches are beingpursued by some groups, others are attempting to develop vaccinestargeting cell-mediated immunity, since evidence show CTLs to beimportant for the control of HIV replication. Phase-I and -IIamulti-epitope vaccine trials have already been conducted with vaccineimmunogens consisting of known CTL epitopes conserved across HIVsubtypes, but have so far fallen short of inducing robust and consistentanti-HIV CTL responses. Thus, a need remains in the art for an effectivevaccine against HIV.

Domestic cats are subject to infection by several retroviruses,including feline leukemia virus (FeLV), feline sarcoma virus (FeSV),endogenous type C oncoronavirus (RD-114), and feline syncytia-formingvirus (FeSFV). Of these, FeLV is the most significant pathogen, causingdiverse symptoms including lymphoreticular and myeloid neoplasms,anemias, immune-mediated disorders, and an immunodeficiency syndromethat is similar to human acquired immune deficiency syndrome (AIDS).Recently, a particular replication-defective FeLV mutant, designatedFeLV-AIDS, has been more particularly associated with immunosuppressiveproperties.

The discovery of feline T-lymphotropic lentivirus (now designated asfeline immunodeficiency virus, FIV) was first reported in Pedersen etal. (1987). Characteristics of FIV have been reported in Yamamoto et al.(1988a); Yamamoto et al. (1988b); and Ackley et al. (1990).Seroepidemiologic data have shown that infection by FIV is indigenous todomestic and wild felines throughout the world. A wide variety ofsymptoms are associated with infection by FIV, including abortion,alopecia, anemia, conjunctivitis, chronic rhinitis, enteritis,gingivitis, hematochezia, neurologic abnormalities, periodontitis, andseborrheic dermatitis. The immunologic hallmark of domestic catsinfected with FIV is a chronic and progressive depletion of feline CD4⁺peripheral blood lymphocytes, a reduction in the CD4:CD8 cell ratio and,in some cases, an increase in CD8-bearing lymphocytes.

Cloning and sequence analysis of FIV has been reported in Olmsted et al.(1989a); Olmsted et al. (1989b); and Talbott et al. (1989). Hosie andJarrett (1990) described the serological response of cats infected withFIV. FIV virus subtypes can be classified according to immunotype basedon the level of cross-neutralizing antibodies elicited by each strain(Murphy and Kingsbury, 1990). Recently, viruses have been classifiedinto subtypes according to genotype based on nucleotide sequencehomology. Although HIV and FIV subtyping is based on genotype (Sodora etal., 1994; Rigby et al., 1993; and Louwagie et al., 1993), little isknown about the correlation between the genotype and immunotype ofsubtypes. FIV viral isolates have been classified into five FIVsubtypes: A, B, C, D, and E (Kakinuma et al., 1995; Yamamoto et al.,2007; Yamamoto et al., 2010). Infectious isolates and infectiousmolecular clones have been described for all FIV subtypes except forsubtypes C and E (Sodora et al., 1994). Subtype C FIV has originallybeen identified from cellular DNA of cats from Canada (Sodora et al.,1994; Rigby et al., 1993; Kakinuma et al., 1995). Examples of FIVstrains identified in the art include (subtype of the strain is shown inparenthesis) Petaluma (A), Dixon (A), UK8 (A), Dutch113 (A), Dutchl9K(A), UK2 (A), SwissZ2 (A), Sendai-1 (A), USCAzepy01A (A), USCAhnky11A(A), USCAtt-10A (A), USCAlemy01 (A), USCAsam-01A (A), PPR (A), FranceWo,Netherlands, Bangston (A/B), Aomori-1 (B), Aomori-2 (B), USILbrny03B(B), TM2 (B), Sendai-2 (B), USCK1gri02B (B), Yokohama (B), USMAsboy03B(B), USTXmtex03B (B), USMCg1wd03B (B), CABCpbar03C (C), CABCpbar07C (C),CABCpady02C (C), Shizuoka (D), Fukuoka (D), LP3 (E), LP20 (E), and LP24(E).

The commercial release of an effective HIV-1 vaccine is not imminenteven after completion of four major phase IIB-III vaccine trials againstHIV/AIDS (Saunders et al. (2012)). Our limited understanding about themechanisms of vaccine protection (Plotkin (2008)) and the identity ofthe protective viral epitopes (Mothe et al. (2011); Koff (2010)) furtherhampers the development of an effective vaccine. Initial studies focusedon antibody-based vaccine designs with an emphasis on generating broadlyvirus neutralizing antibodies (bNAbs) (Stamatatos (2012)). However, twophase-III vaccine trials using envelope (Env) immunogens failed (Flynnet al. (2005); Pitisuttithum et al. (2006)). Subsequent focus was placedon the T-cell-based vaccines that generate protective cell-mediatedimmunity (CMI) against global HIV-1 isolates (Buchbinder et al. (2008)).The CMI responses, essential for an effective vaccine, most likelyinclude cytotoxic T lymphocyte (CTL) activities that specifically targetHIV-1 infected cells (Ogg et al. (1998); Walker et al. (1988); Belyakovet al. (2012)). Unlike NAb epitopes which reside exclusively on the Envproteins, the selection of specific vaccine epitopes for the developmentof T-cell-based vaccines is more difficult to achieve. A vast number ofCTL-associated epitopes can be found to span the whole length of mostHIV proteins (Los Alamos National Laboratory (LANL) database,hiv-web.lanl.gov/content/immunology/maps/maps.html) (Llano et al.(2009)). The goal to develop T-cell-based vaccines is challenged by thecapacity of the virus to evade antiviral immunity through mutation(s)for resistance (Li et al. (2011); Leslie et al. (2004)).

A recent phase III trial consisting of priming with a gag-pr-gp41-gp120canarypox vectored vaccine and boosting with Env gp120 induced bothhumoral immunity and CMI and conferred a modest overall efficacy(Rerks-Ngarm et al. (2009)). However, phase I and II vaccine trialsconsisting of cross-subtype conserved CTL-associated peptide epitopeshave shown minimal CMI responses (Sanou et al. (2012a); Hanke et al.(2007); Salmon-Ceron et al. (2010)). Therefore, a thorough selection ofpotent anti-HIV T cell-associated epitopes, which are conserved amongHIV-1 subtypes and do not mutate without negatively affecting viralfitness (Troyer et al. (2009); Goulder et al. (2008); Rolland et al.(2007)), would be valuable for an effective HIV-1 vaccine. One approachis to select conserved, non-mutable CTL epitopes on essential viralstructural proteins or enzymes that also persist on the older subgenusesof the lentivirinae which have survived evolutionary pressure (Yamamotoet al. (2010)). Such an approach was successfully used in thedevelopment of the initial smallpox vaccines (Jenner (1798)). In linewith this strategy, the recognition of conserved epitopes on otherlentivirus species has been made by the PBMC from HIV-1 positive (HIV+)humans (Balla-Jhagjhoorsingh et al. (1999)), HIV-2 vaccinated andSIV-challenged non-human primates (Walther-Jallow et al. (2001)), andHIV-1 p24-vaccinated and FIV-challenged cats (Abbott et al. (2011);Coleman et al. (2005)).

The viral enzyme, reverse transcriptase (RT), is one of the mostconserved viral proteins by possessing the lowest entropy value amongthe HIV-1 proteins from various subtypes (Yusim et al. (2002)) andcontains many CTL-associated epitopes (Walker et al. (1988)). The RTproteins of HIV-1 and FIV also share the highest degree of identity intheir amino acid (aa) sequences (Yamamoto et al. (2010)). The currentstudies were undertaken to identify the conserved CTL-associatedepitopes on FIV and HIV-1 RT proteins which are recognized by the PBMCand T cells from HIV+ subjects. The major objective of such studies isto identify evolutionarily-conserved CMI epitopes that may be moreresistant to mutation, and thus useful in the development of aneffective, T-cell-based HIV-1 vaccine.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns methods and materials for inducing animmune response in an animal or person against an immunodeficiencyvirus, such as HIV, SIV, or FIV. In one embodiment, a method of theinvention comprises administering one or more antigens and/or immunogensto the person or animal wherein the antigen and/or immunogen comprisesone or more evolutionarily conserved epitopes of immunodeficiencyviruses. In one embodiment, the epitope is one that is conserved betweenHIV and SIV, or between HIV and FIV. In another embodiment, the epitopeis one that is conserved between HIV, SIV, and FIV. In one embodiment,the epitope is a T-cell epitope. In a specific embodiment, the T-cellepitope is a cytotoxic T lymphocyte (CTL) and T-helper (Th) epitope.

The subject invention also concerns evolutionarily conserved epitopes ofimmunodeficiency viruses. In one embodiment, the epitope is one that isconserved between HIV and SIV, or is one that is conserved between HIVand FIV. In another embodiment, the epitope is one that is conservedbetween HIV, SIV, and FIV. In one embodiment, the epitope is a T-cellepitope. In a specific embodiment, the T-cell epitope is a cytotoxic Tlymphocyte (CTL) and T-helper (Th) epitope.

The subject invention also concerns antibodies that bind to HIV, SIV,and/or FIV epitopes. In one embodiment, an antibody of the inventionbinds specifically to an HIV protein, e.g., an HIV p24 protein. Inanother embodiment, an antibody of the invention binds specifically toan FIV protein, e.g., an HIV p24 protein. In a further embodiment, anantibody of the invention binds specifically to both an HIV and an FIVprotein, i.e., the antibody cross-reacts with both an HIV and an FIVprotein, such as a p24 protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent and Trademark Officeupon request and payment of the necessary fee.

FIGS. 1A and 1B. NetCTL-1.2 prediction of HIV, SIV, and FIV CTLepitopes.NetCTL-1.2, which is based on proteosomic C-terminal cleavage,TAP transport efficiency, and epitope binding to MHC class I alleles,was used to predict CTL epitopes shown by HLA supertypes(cbs.dtu.dk/services/NetCTL/). The total number of predicted epitopes byHLA supertype (FIG. 1A): HIV (78), SIV (74), and FIV (85) were talliedafter analysis of the full-length integrase sequence from each virus.The predicted CTL epitopes were compared and the conserved epitopesbetween the viruses were identified based on aa position and samepredicted HLA supertype (FIG. 1B): HIV-SIV (34), HIV-FIV (25), andHIV-SIV-FIV (17).

FIG. 2. Possible Location of Counterpart Epitopes. HIV proteins (A, B)aligned to FIV proteins (A, B) showing four HIV epitopes (h1, h2, h3,h4) and three FIV epitopes (f1, f2, f3) with arrows indicating thelocation of the direct counterpart (arrow a) and indirect counterpartepitopes (arrows b, and c).

FIGS. 3A and 3B. Comparison Between FIV RT and HIV-1 RT. CD3+CD4+ andCD3+CD8+ T-cell CFSE-proliferation (n=24) and PBMC IFNγ ELISpot (n=28)responses of HIV+ subjects to overlapping FIV RT (FRT 1-21, top) andHIV-1 (HRT 1-21, bottom) peptide pools. Results are shown as a meanvalue of % CFSE_(low) or spot forming unit (SFU) per 10⁶ PBMC withpercentage of responders over total subjects tested (% Responder).Uninfected subjects (n=10) had negligible to no responses. Dark red boxrepresents the highest response while boxes with lighter shades of redshowing lesser responses with blue representing minimal to no responses.Thresholds are ≧2% for CFSE-proliferation and ≧70 SFU for ELISpot.

FIGS. 4A and 4B. Comparison Between FIV p24 and HIV-1 p24. CD3+CD4+ andCD3+CD8+ T-cell CFSE-proliferation (n=24) and PBMC IFNγ ELISpot (n=31)responses to overlapping FIV p24 (Fp 1-17, top) and HIV-1 p24 (Hp 1-18,bottom) peptide pools. Results are shown as a mean value of % CFSE_(low)or spot forming unit/10⁶ PBMC with (% Responder). Dark red boxrepresents the highest response while lighter shades of red show lesserresponses with blue representing minimal to no response.

FIG. 5. Sequence comparison between HIV-1_(LAI) reverse transcriptase(RT) (441 aa) and FIV_(FC1) RT (445 aa).

FIG. 6. Sequence comparison between HIV-1_(LAI) RT (441 aa) andSIV_(Mm251) (439 aa).

FIG. 7. Sequence comparisons between FIV-Pet p24 (223 aa) and SIV-DeltaB670 p24 (228 aa), between HIV1 HXB2 p24 (231 aa) and FIV-Pet p24 (223aa), and between HIV1-HXB2 p24 (231 aa) and SIV-Delta B670 p24 (228 aa).

FIGS. 8A and 8B. Chimera HIVp24/FIV-Shizuoka infected PBMC (batch 10)with MAb reactive to both FIV p24 and HIV-1 p24 (FIG. 8A). ChimeraHIVp24/FIV-Shizuoka infected PBMC (batch 10) with MAb reactive to FIVgp95 (surface envelope) (FIG. 8B). Chimera HIVp24/FIV-Shizuoka infectedPBMC (batch 10) with isotype IgG control antibody (FIG. 8C). Indirectfluorescent antibody (IFA) analysis of feline PBMC infected with chimeraHIVp24/FIV-Shizuoka (subtype-D backbone) virus. Note HIV-1_(UCD1)belongs to HIV-1 subtype B. The culture supernatant from chimeratransinfected 293T cells was inoculated into uninfected feline PBMCculture, and then 2-3 weeks later when the cells were dying co-culturedwith fresh uninfected feline PBMC for 2-3 weeks before the cells wereused for IFA analysis. Murine MAb reactive to both FIV p24 and HIV-1 p24(FIG. 8A) and anti-FIV gp95 (surface envelope) MAb (FIG. 8B) incombination with FITC-labeled anti-mouse IgG detect chimeraHIVp24/FIV-Shizuoka virus infected PBMC (green fluorescent cell in FIGS.8A and 8B). Murine isotype IgG control antibody in combination withFITC-labeled anti-mouse IgG has no fluorescent reactivity (FIG. 8C).Thus, the chimera HIVp24/FIV-Shizuoka virus is infectious to felinePBMC.

FIGS. 9A-9D. Indirect fluorescent antibody (IFA) analysis of feline PBMCinfected with chimera HIVp24/FIV-Petaluma (subtype-A backbone) virus.Chimera HIVp24/FIV-Petaluma infected PBMC (batch 14) with MAb reactiveto both FIV p24 and HIV-1 p24 (FIG. 9A). Chimera HIVp24/FIV-Petalumainfected PBMC (batch 14) with anti-FIV gp95 MAb (FIG. 9B). ChimeraHIVp24/FIV-Petaluma infected PBMC (batch 14) with anti-FIV gp95 MAb(FIG. 9C). Chimera HIVp24/FIV-Petaluma infected PBMC (batch 14) withisotype IgG control antibody (FIG. 9D). The culture supernatant fromchimera transinfected 293T cells was inoculated into uninfected felinePBMC culture, and then 2-3 weeks later when the cells were dyingco-cultured with fresh uninfected feline PBMC for 2-3 weeks before thecells were used for IFA analysis. Murine MAb reactive to both FIV p24and HIV-1 p24 (FIG. 9A) and anti-FIV gp95 (surface envelope) MAb (FIGS.9B and 9C) in combination with FITC-labeled anti-mouse IgG detectchimera HIVp24/FIV-Shizuoka virus infected PBMC (green fluorescent cellin FIGS. 9A, 9B, 9C). Murine isotype IgG control antibody in combinationwith FITC-labeled anti-mouse IgG has no fluorescent reactivity (FIG.9D). Thus, the chimera HIVp24/FIV-Petaluma virus is infectious to felinePBMC.

FIG. 10. Reactivity of MAbs to HIV-1 and FIV p24 proteins in Westernblot (WB) with FIV (top) or HIV-1 (bottom) p24 substrate. FIVp24-specific MAbs HL2350 and HL2351 do not cross react to HIV-1 p24proteins or its degraded proteins in WB and ELISA. HIV-1 p24-specificMAbs HL2309 and HL2310 do not cross react with FIV p24 proteins or itsdegraded proteins in WB and ELISA.

FIGS. 11A and 11B. Reactivity of HIV-1+ human subjects: comparisonbetween FIV RT and HIV-1 RT. CD3+CD4+ and CD3+CD8+ T-cellCFSE-proliferation (n=24) and PBMC IFNγ ELISpot (n=28) responses of HIV+subjects to overlapping FIV RT (FRT 1-21, top) and HIV-1 (HRT 1-21,bottom) peptide pools. Results are shown as a mean value of % CFSE_(low)or spot forming unit (SFU) per 10⁶ PBMC with percentage of respondersover total subjects tested (% Responder). Uninfected subjects (n=10) hadnegligible to no responses. Dark red box represents the highest responsewhile boxes with lighter shades of red showing lesser responses withblue representing minimal to no responses. Thresholds are ≧2% forCFSE-proliferation and ≧70 SFU for ELISpot.

FIGS. 12A and 12B. Reactivity of HIV-1+ human subjects: comparisonbetween FIV p24 and HIV-1 p24. CD3+CD4+ and CD3+CD8+ T-cellCFSE-proliferation (n=24) and PBMC IFNγ ELISpot (n=31) responses tooverlapping FIV p24 (Fp 1-17, top) and HIV-1 p24 (Hp 1-18, bottom)peptide pools. Results are shown as a mean value of % CFSE_(low) or spotforming unit/10⁶ PBMC with (% Responder). Dark red box represents thehighest response while lighter shades of red show lesser responses withblue representing minimal to no responses. HIV p24 sequence is longerthan that of FIV. Therefore, the alignment shifts after Fp6, and Fp7 isthe counterpart of Hp8 and so on.

FIG. 13A. Full aa sequence alignment of HIV-1_(LM) reverse transcriptase(RT) (441 aa) and FIV_(FC1) RT (445 aa) show 47.7% identity and 70.8%homology. Based on the results in FIGS. 11A and 11B above, four epitopicregions (HRT3/FRT3, HRT6/FRT6, HRT11/FRT11, HRT13/FRT13) with moderateto high reactivity to FIV RT peptide pool by either IFNγ-ELISpot orCFSE-proliferation were selected for aa sequence analysis usingGENESTREAM network server. Red aa sequence represents HRT peptide poolsequence, while blue aa sequence represents FRT peptide pool sequence.The aa sequence with red or blue underline are counterpart sectionbetween FIV and HIV RT proteins and are also evaluated for aa identityand homology as shown on the right. GENESTREAM network server(xylian.igh.cnrs.fr/bin/align-guess.cgi): Pearson, W. R., Wood, T.,Zhang, Z., and Miller, W. (1997), Comparision of DNA sequences withprotein sequences, Genomics 46: 24-36.

FIG. 13B. Full aa sequence alignment of HIV-1_(LAI) RT (441 aa) andSIV_(Mm251) (439 aa) show 59.6% identity and 81.9% homology. Based onthe results in FIG. 14 below, four epitopic regions (HRT3, HRT6, HRT11,HRT14) with moderate to high reactivity to HIV RT peptide pool byIFNγ-ELISpot were selected for aa sequence analysis using GENESTREAMnetwork server. Red aa sequence represents HRT peptide pool sequence,while blue aa sequence represents FRT peptide pool sequence. Thesecounterpart peptide pool regions of HIV and SIV RT proteins are alsoevaluated for aa identity and homology as shown on the right. The yellowhighlight represents the HIV peptide pool and the macaques (immediatelybelow the counterpart SIV sequence) reacting to the correspondingpeptide pool. PBMCs from infected macaques were also tested with shorterpeptides of HRT3 which are known to be CTL epitope for HIV+ humans.PBMCs from macaques R395 and R397 reacted to peptide “WRKLVDFRE” (SEQ IDNO:450) (red & counterpart blue underlined) while that of macaque R416reacted to both overlapping peptide pool HRT3 and individual peptide“KWRKLVDFRELNKR” (SEQ ID NO:166) in green highlight. Furthermore, PBMCof macaque R422 reacted to FIV p24 peptide “NPWNTPVFAIKKK” (SEQ IDNO:61) and its SIV counterpart sequence is shown in aqua highlight. Thereason why only R416 responded to overlapping peptide pool while others(R395, R397, R422) only reacted to smaller peptides are technicalreasons such as smaller peptides can bind to MHC more readily than thelarger peptides (11-16mers) used for overlapping peptide pools and thatother peptide(s) in the pool can decrease the responses.

FIGS. 14A-14B. IFNγ ELISpot Responses to HIV RT (reverse transcriptase)and HIV p24 (core protein) of the Primate PBMCs. Overlapping HIV-1 p24(FIG. 14A) and RT (FIG. 14B) peptide pool analyses are shown for nineSIV-infected rhesus macaques and four pre-infection macaques. FrozenPBMC were thawed and plated at the concentration of 1.4×10⁵ viable cellsper mL. Peptides were used at a concentration of 15 μg/mL. Each barrepresents an individual primate's response in spot forming units(SFU/10⁶ PBMC) after subtraction of 2 times the media control; exceptfor the black and red bars. The black bar represents the averageresponse of the pre-infection responders (Av. n=3) and the red barrepresents the average response of all 4 pre-infection samples (Av.total n=4). Since these cells were frozen for over 5 years, positiveresponses are values of ≧50 SFU. We believe that fresh(non-cryopreserved) cells will give higher responses to HIV p24 peptidepools (FIG. 14A: Hp1-Hp18) and HIV RT peptide pools (FIG. 14B:Hr1-Hr21). Various mitogens (Mito.) (concanavalin A, Staphyloccocalenterotoxin A, phytohemaglutinin A) were used since these frozen cellsdid not always respond to mitogen.

FIGS. 15A and 15B. HIV-1 and FIV p24 sequences and individual peptidesin the peptide pools. (SEQ ID NOs:257-262,264-319,321-335,337-370).

FIG. 16. FRT3 induces the secretion of multiple cytokines in the PBMC of3/5 HIV+ individuals tested.

FIGS. 17A-17F. IFNγ and CD8+ T cell proliferation responses ofHIV-infected subjects to HIV and FIV reverse transcriptase (RT) peptidepools. The IFNγ ELISpot (FIGS. 17A and 17B, n=32; 12 LTS, 12 ST, 8 ART+)and CD3+CD8+ T-cell proliferation (FIGS. 17C and 17D, n=26; 11 LTS, 7ST, 8 ART+) responses to overlapping peptide pools of HIV RT (H1-H21;FIGS. 17A and 17C) and FIV RT (F1-F21; FIGS. 17B and 17D) are shown. TheHIV+ subjects (panel-A insert for FIGS. 17A-17D) consisted of long-termsurvivors (LTS) who have had HIV infection for over 10 years withoutantiretroviral therapy (ART) (LTS; black bar); subjects recentlydiagnosed with short-term infection without ART (ST; grey bar); andsubjects on ART at various duration of infection (ART+, red bar). Eachbar represents a positive response by an individual with a threshold of70 spot forming units (SFU) per 10⁶ PBMC for ELISpot or threshold of 3%CFSElow for CD3+CD8+ T-cell proliferation. Cells from each individualwere stimulated with T-cell mitogen, phytohemaglutinin A (PHA), aspositive control. The HIV-control subjects (n=10) had no responses (datanot shown). All responses below the positive threshold are not shown toclearly distinguish those positive values close to the threshold.

The average frequencies of IFNγ and proliferation responders to HIV-1(FIG. 17E) and FIV (FIG. 17F) RT peptide pools are derived from FIGS.17A-17D and are shown as % responders. The solid bar for each peptiderepresents an average responder frequency of IFNγ responses, while thegrey bar represents of the CD8+ T-cell proliferation responses.

FIGS. 18A-18B. CD3+CD4+ T-cell proliferation responses to HIV and FIV RTpeptide pools. The CD3+CD4+ T-cell proliferation responses tooverlapping peptide pools of HIV RT (H1-H21; FIG. 18A) and FIV RT(F1-F21; FIG. 18B) are shown for HIV+ subjects (n=26; 11 LTS, 7 ST, 8ART+). The insert in panel FIG. 18A shows the bar color codes for bothpanels as: LTS without ART (LTS; black bar), ST without ART (ST; greybar), and those on ART (ART+; white bar). Each bar represents a positiveresponse by an individual with a positive threshold of 70 SFU per 10⁶PBMC for ELISpot or 3% CFSE^(low) for CD3+CD4+ T-cell proliferation.None of the HIV− control subjects (n=10) had positive responses (datanot shown). Responses below positive thresholds are not shown.

FIGS. 19A-19B. Persistence of IFNγ and CD8+ T-cell proliferationresponses to selected peptide pools. The IFNγ (FIG. 19A) and CD8+ T-cellproliferation (FIG. 19B) responses of HIV+ subjects who responded firsttime (t1) and second time (t2, at least 1 year later) are shown forpeptide-pool F3 (both analyses), H11 (both analyses), and H6 (IFNγ) orF6 (T-cell proliferation). The total number of HIV+ subjects whoparticipated is 22 subjects (FIG. 19A: 9 LTS, 8 ST, 5 ART+) in IFNγstudy and 11 subjects (FIG. 19B: 3 LTS, 6 ST, 2 ART+) in CD8+ T-cellproliferation study. However, the number of subjects with differentclinical status differs among the peptide-pool groups since it is basedon the number of responders to the peptide at the first time (t1). Inaddition, the IFNγ responses to H6 and F3 have a third time point (t3,≧2 yr). The p-value of each peptide-pool group indicates that theresults from t1 are statistically different from those from t2 whenp<0.5. Only CD8+ T-cell proliferation responses to F6 at t1 arestatistically different from those at t2. A statistical comparisonsbetween t2 and t3 of H6 (n=5) and F3 (n=5) were p=0.124 and p=0.133,respectively (p-value not shown).

FIGS. 20A-20B. F3 peptide epitopes recognized by F3 responders. Thepeptide-pool F3 consists of five overlapping 13-15mer peptides spanningfrom amino- to carboxyl-terminal (F3-1, F3-2, F3-3, F3-4, and F3-5).IFNγ (FIG. 20A; n=10; 5 LTS, 3 ST, 2 ART+) and CD8+ T-cell proliferation(FIG. 20B; n=8; 3 LTS, 3 ST, 2 ART+) by cells from HIV-infected F3responders to each of these peptides are shown along with responses toF3 pool. F3 responders consist of those subjects with long-term HIV-1infection but not on ART (LTS; black bar); those with short-terminfection and not on ART (ST; grey bar); and those on ART with variousduration of infection (ART+; red bar). All responses below positivethresholds are not shown.

FIGS. 21A-21D. Characterization of CTL-associated epitopes on H6, H11,F3, and F6 pools. ICS analysis for perforin (Perf), granzyme A (GrzA),and granzyme B (GrzB) expression is shown for CD8+ T cells (left column)and CD4+ T cells (right column) from selected HIV+ responders ofdesignated peptide pools (H6, n=8; H11, n=8; F3, n=11; F6, n=6; and PHA,n=12) (FIGS. 21A-21C). The HIV+ subjects (panel-A insert for FIGS.21A-21C) consist of the following individuals: those with long-terminfection without ART (LTS; black closed circle); those recentlydiagnosed, with short-term infection without ART (ST; grey closedcircle); and those on ART with various duration of infection (ART+; opencircle). The number of each clinical status group is the following foreach peptide-pool group: H6 (4 LTS, 2 ST, 2 ART+), H11 (4 LTS, 1 ST, 3ART+), F3 (5 LTS, 3 ST, 3 ART+), F6 (2 LTS, 2 ST, 2 ART+), and PHA (5LTS, 3 ST, 4 ART+). Six F3-pool responders (5 LTS, 1 ART+) were testedfor Perf (•), GrzA (□), and GrzB (Δ) responses to the five 13-15mer F3peptides (FIG. 21D). Only three subjects were the same as those fromabove (FIGS. 21A-21C), but the blood was collected at a differenttime-point.

FIGS. 22A-22B. The aa sequence identity between counterpart HIV/FIVpeptide pools and between various lentiviruses. The percentage of aaidentity (FIG. 22A) between the sequences of each HIV (H) peptide pooland its counterpart FIV (F) peptide pool were obtained by alignment ofthe sequences using ebi.ac.uk/Tools/psa/emboss_needle/. Note that thehighest aa identity observed is 68.7% with peptide-pools H4/F4 and thesecond highest is 66.7% with peptide-pools H3/F3. In FIG. 22B, thepercentages shown on the right of the diagonal divider represent % aasequence similarity and those on the left represent % aa sequenceidentity between the two viruses intersecting the value. The lentivirusstrains compared are HIV-1HXB2 (GenBank K03455.1), FIVFC1 (DQ365597.1),SIVMm251 (AAB59906.1), and CAEV (AAG48629.1).

FIGS. 23A-23C. IFNγ responses to HIV, FIV and SIV p24 peptide pools. TheIFNγ ELISpot responses to overlapping peptide pools of HIV p24(Hp1-Hp18, n=31; FIG. 23A), FIV p24 (Fp1-Fp17, n=31; FIG. 23B), and SIVp24 (Sp1-Sp17, n=15; FIG. 23C) are depicted as spot forming units (SFU)per 10⁶ PBMC. The HIV⁺ subjects (FIG. 23A insert for FIGS. 23A and 23B;FIG. 23C insert for FIG. 23C) consisted of long-term survivors (LTS)without antiretroviral therapy (ART) (LTS; black bar); recentlydiagnosed subjects (<1 year) with short-term infection without ART (ST;grey bar); and those receiving ART with various duration of infection(ART+, red bar). Each bar represents a positive response by anindividual subject with a threshold of >50 SFU per 10⁶ PBMC. Asteriskafter the peptide pool(s) represent those with highest frequency ofresponders. The FIV p24 pool with the highest and the most frequentresponses and its counterpart HIV-1 and SIV pools are highlighted inblue.

FIGS. 24A-24F. Proliferation responses to HIV, FIV and SIV p24 peptidepools. The CD3⁺CD8⁺ T-cell proliferation responses to overlappingpeptide pools of HIV p24 (Hp1-Hp18, n=27; FIG. 24A), FIV p24 (Fp1-Fp17,n=27; FIG. 24B), and SIV p24 (Sp1-Sp17 n=13; FIG. 24C) are depicted as %CFSE^(low) for CD3⁺CD8⁺ and CD3⁺ CD4⁺ T-cell proliferation. The HIV⁺subjects (FIG. 24A insert for FIGS. 24A and 24B; FIG. 24C insert forFIG. 24C) consisted of long-term survivors (LTS) without ART (LTS; blackbar); recently diagnosed subjects (<1 year) with short-term infectionwithout ART (ST; grey bar); and those on ART with various duration ofinfection (ART⁺, red bar). Each bar represents a positive response by anindividual with a threshold of >1% CFSE^(low) for CD3⁺CD8⁺ T-cellproliferation. Asterisk after the peptide pool(s) represents those withhighest frequency of responders. The two FIV p24 pools with the highestand the most frequent responses and their counterpart HIV-1 and SIVpools are highlighted either in blue (Hp15/Fp14/Sp14) or red(Hp10/Fp9/Sp9).

FIGS. 25A-25B. The persistence of IFNγ and T-cell proliferationresponses to selected peptide pools. The IFNγ (FIG. 25A) and CD8⁺ T cellproliferation (FIG. 25B) responses of HIV⁺ subjects who responded at thefirst sample collection (t1), 2 yr later (t2), and 4 yr later (t3) areshown for peptide pool Fp14 (IFNγ), Hp15 (IFNγ), Hp10 (proliferation),and Fp9 (proliferation). The p-value indicates statistical differencesbetween t1 and t2, t2 and t3, and t1 and t3. The threshold for IFNγ andproliferation responses are >50 SFU and >1% CFSE^(low), respectively.

FIGS. 26A-26C. IFNγ and CD8⁺ T-cell proliferation responses to Fp9,Fp14, and Hp15 peptide epitopes. Peptide pool Fp14 consists of fouroverlapping 13-15mer peptides spanning the amino- to carboxyl-terminus(Fp14-1, Fp14-2, Fp14-3, Fp14-4), while pools Hp15 (Hp15-1, Hp15-2,Hp15-3) and Fp9 (Fp9-1, Fp9-2, Fp9-3) each consist of three overlapping13-15mer peptides. IFNγ responses to individual Fp14 peptides (FIG. 26A;n=9), Hp15 peptides (FIG. 26B; n=9) and CD8⁺ T-cell proliferationresponses to individual Fp9 peptides (FIG. 26C; n=7) are shown alongwith responses to their corresponding peptide pools. Only responders topools Fp14 (FIG. 26A), Hp15 (FIG. 26B), and Fp9 (FIG. 26C) were tested.The responders consisted of long-term survivors (LTS) without ART (LTS;black bar); recently diagnosed subjects (<1 year) with short-terminfection without ART (ST; grey bar); and those on ART with variousduration of infection (ART⁺, red bar). Each bar with (*) in panels A andB are from the same three LTS.

FIGS. 27A-27F. IFNγ and T-cell proliferation responses to 9-13mers ofFIV peptides Fp9-3, Fp14-3, and Fp14-4. Six 9-12mer peptides for the Fp9pool (FIGS. 27A, 27C, 27E) and seven 9-13mer peptides for the Fp14 pool(FIGS. 27B, 27D, 27F) described in Table 13 were tested for theirability to induce CD8⁺ T-cell proliferation (FIGS. 27A and 27B), CD4⁺T-cell proliferation (FIGS. 27C and 27D), and IFNγ production (FIGS. 27Eand 27F) and compared to results with the Fp9 and Fp14 peptide pools.13-15mer peptides Fp9-3, Fp14-3, and Fp14-4; and mitogen (PHA) wereincluded as controls. A total of nine HIV⁺ responders to the Fp9 pool(FIGS. 27A, 27C, 27E) and 10 HIV⁺ responders to the Fp14 pool (FIGS.27B, 27D, 27F) were tested up to 4 yr after the beginning of the study.Two to four of the responders were lost to follow up. Theirproliferation and IFNγ results to the 13-15mer peptides Fp9-3, Fp14-3,and Fp14-4, and are denoted with an (*) for each individual missing.

FIGS. 28A-28D. Stimulation of cytotoxins by CTL epitopes on Fp9, Fp14,and Hp15 peptide pools, and their individual peptides. The ICS analysesfor perforin (Perf, ∘), granzyme A (GrzA, □), and granzyme B (GrzB, Δ)are shown for CD8⁺ T cells (FIGS. 28A, 28D) and CD4⁺ T cells (FIGS. 28B,28C) from five HIV⁺ responders with or without ART. Two LTS/ART⁻, oneLTS/ART⁺, one ST/ART⁺, and one ST/ART⁻ were first evaluated (FIGS. 28A,28B). Peptides tested included Fp9, Fp14, and/or Hp15 peptide pools orlarge 13-15mer peptides (FIGS. 28A, 28B). Responses from five HIV⁺responders of short-term HIV-infected subjects not on ART (ST/ART⁻) weretested using small 9-13mer peptides within Fp9-3, Fp14-1, Fp14-3,Fp14-4, and Sp14-1 (FIG. 28C, 28D). Note that Sp14 pool is a single13mer peptide Sp14-1. Counterpart peptides are shown with blue-dashedbox for Fp14-1a/Hp15-1c/Sp14- 1c, purple-dashed box forFp14-1b/Hp15-1a/Sp14-1b, and red-dashed box forFp14-3/4f/Hp15-2/3a/Sp14-1a. The peptides tested with one less subjectare denoted by (**), while their number of subjects and number ofresponses are denoted by (*). Each separate symbol color represents onesubject, and each color-coded subject is shown with his/her infectionstatus. The threshold for T cells expressing cytotoxin is set at >0.1%CD4⁺ or CD8⁺ T cells.

FIG. 29. Summary of functional epitope analyses. Results from threefunctional analyses are summarized according to frequency of HIV⁺ (lanes1-4) or HIV⁻ (lane 5) responders and are shown as (+) for frequencyof >25% responders, (±) for 19-25% responders, (−) for <19% responders,or as (*) for not available. Each lane, abridged in the insert, showsthe ability of the peptide pool or peptide to stimulate an IFNγ response(lane 1), CD8⁺ T-cell proliferation (lane 2), and CD4⁺ T-cellproliferation (lane 3). Lane 4 denotes the ability to inducecytotoxin(s) in only CD8⁺ T cells (+) or in both CD8⁺ and CD4⁺ T cells(++) by four or more HIV⁺ subjects when at least nine subjects tested.In lane 5, the positive result (+) indicates substantial frequency ofproliferation responses from HIV subjects and may indicate a safetyconcern; whereas a negative result (−) indicates no substantial HIV⁻response to the peptide pool or peptide. Positive response of HIVcontrol had CD8⁺ T-cell proliferation response in 30-42% of HIV⁻subjects. The large 13-15mer peptides with the best CMI responseswithout stimulation in HIV subjects are shown with dashed boxes, whilethe best small peptides are shown with solid boxes. Therefore, Hp15-1,Hp15-2/3a, Fp14-4, Fp14-1b, Fp14-3/4e, and Fp14-3/4f peptides appear tocontain the best potential epitopes to target for use as HIV immunogens.

FIGS. 30A-30F. CD8⁺ T-cell proliferation (FIGS. 30A-30C) and IFNγ (FIGS.30D-30F) responses to HIV-1, FIV, and SIV p24 peptide pools by HIV⁺subjects (Roff et al. 2015). The PBMCs were stimulated with HIV-1(Hp1-Hp18) (FIGS. 30A, 30D) and corresponding SIV (Sp1-Sp17) (FIGS. 30C,30F) and FIV (Fp1-Fp17) (FIGS. 30B, 30E) p24 peptide pools at 5 μg/mLpeptide pool for proliferation and 6-8 μg pool/well for IFNγ. Positivecontrol cultures were stimulated with T-cell mitogen phytohemaglutinin A(PHA) or concanavalin A (ConA) each at 5 μg/mL. The PBMCs and T cellswere from long-term survivors (LTS, black bar), subjects with short-terminfection without ART (ST, grey bar), and subjects on ART at variousduration of infection (ART⁺, red bar). The results are shown aftersubtraction of the individual media control and responses to eachpeptide pool by PBMC or T cells from HIV-negative subjects. The HIVcounterpart for FIV and SIV pools is offset by one additional poolstarting from Fp7/Sp7 and therefore HIV counterpart for Fp9/Sp9 andFp14/Sp14 are Hp10 and Hp1, respectively. They are shown with red andblue boxes for Hp10/Fp9/Sp9 and Hp15/Fp14/Sp14, respectively.

FIGS. 31A and 31B. Stimulation of cytotoxins by CTL epitopes on Fp9,Fp14, and Hp15 peptide pools, and their individual peptides by HIV⁺subjects. The intracellular cytotoxin staining (ICS) analyses forperforin (Perf, ∘), granzyme A (GrzA, □), and granzyme B (GrzB, Δ) areshown for CD8⁺ T cells (FIG. 31A) and CD4⁺ T cells (FIG. 31B) from fiveHIV⁺ responders with or without ART. Two LTS/ART⁻, one LTS/ART⁺, oneST/ART⁺, and one ST/ART⁻ were first evaluated (FIGS. 31A, 31B). Peptidestested included Fp9, Fp14, and/or Hp15 peptide pools or large 13-15merpeptides (FIGS. 31A, 31B). Each separate symbol color represents onesubject, and each color-coded subject is shown with his/her infectionstatus. The threshold for T cells expressing cytotoxin is set at >0.1%CD4⁺ or CD8⁺ T cells. Additional five ST/ART⁻ subjects are shown in FIG.6 of reference (Roff et al. 2015).

FIGS. 32A and 32B. T-cell proliferation responses to FIV p24 peptidepools by vaccinated cats. CD8⁺CD4⁺ (FIG. 32A) and CD3⁺CD8⁺ (FIG. 32B)T-cell proliferation responses are shown for prototype FIV-vaccinatedcats. The FIV vaccinated cats were semi-inbred cats with blue, dark red,and pink/green bars from different MHC-lineage colonies. The semi-inbredcats with light pink and green bars are from the same MHC lineage. PoolsFp9 and Fp14 are recognized by both HIV⁺ subjects and vaccinated cats(i.e., evolutionarily conserved [EC] epitopes) but pools Fp3 and Fp4 arenot recognized by HIV⁺ subjects (i.e., non-EC epitopes). The IFNγresponses of these cats were either low or below the cut-off thresholdof 50 SFU/1×10⁶ PBMC. The peptides that stimulated the mostproliferation responses: non-EC peptide Fp4-3 in pool Fp4 and EC peptideFp14-1 in pool Fp14 were used in in vivo study below (FIG. 34).

FIGS. 33A-33C. IFNγ and T-cell proliferation responses to Fp9 and Fp14by the FIV-vaccinated cats and FIV-infected cats. The T cells or PBMCfrom 32 FIV-vaccinated cats were stimulated with either Fp9 or Fp14 andevaluated for proliferation (FIG. 33A) or IFNγ (FIG. 33B) responses. Inaddition, the PBMC from 32 FIV-infected cats at 22-52 weeks of infectionand 20 HIV-1 p24-immunized cats were tested for IFNγ responses to thesame peptide pools (FIG. 33B). Three types of laboratory cats were usedin these studies: semi-inbred line 1 (Semi-Inbred 1), semi-inbred line 2(Semi-Inbred 2), and outbred cats (Outbred) from laboratory cat vendors.Semi-inbred cats have been inbred for 3-5 generations with definedfeline leukocyte antigen (FLA) alleles and are divided into two FLAhaplotype lines. FIV vaccine consisted of prototype dual-subtype FIVvaccine (Coleman et al. 2014). The insert in each panel defines thenumbers of each type of cats as well as vaccination (Vac.) or infection(Infect.) status. The number of cat responders used varies between theanalyses performed for the individual Fp14 peptides (FIG. 33C, left forIFNγ and right for proliferation).

FIG. 34. 3 MAPs with 1-2 FIV peptides for Table 17. These peptidesstimulate IL-2 and IFNγ responses in T cells from FIV-vaccinated cats.Two different formulations of lipophylic (palmitate C16, PAM) MAP weremixed in FD-1 adjuvant and immunized in prototype vaccine-primed SPFcats. Furin-sensitive sequence (RVKR) (SEQ ID NO:494) was used to linkthe two peptides (Nakayama 1997; Kotterman and Schaffer 2014). Afterentering the cell, the peptide chains can enter the endoplasmicreticulum and trans-Golgi network, where the furin processes the furinsensitive sequence into two peptides. Subsequently, these peptides bindto MHC. The peptide-MHC complexes are then transported to the cellsurface where they interact with T cells.

FIG. 35. FIV-enhancing versus inhibitory epitopes on peptides and MAPsused in pilot MAP Study. Statistically significant enhancement (Fp4-3,FRT3-3, MAP1) and inhibition (FRT3-4, MAP2) of in vitro FIV infection inthe PBMC from naïve cats are shown. Virus+ control, FIV-enhancingmitogen (ConA) control, and uninfected cell control had an average RTtiter (cpm/mL) of 26,518 (1:500 dilution), 54,634 (1:10000) and 2612(1:500), respectively. Abbreviation: MAP1 plus MAP2 mix (MAP1+2).

FIGS. 36A and 36B. FIV/HIV-1 enhancing versus inhibitory epitopes.Statistically significant enhancement (Fp4-3 & FRT3-3) and inhibition(Fp9-3 & FRT3-4) of FIV infection of naïve cat PBMC were observed (FIG.36A). Virus positive (+) control, enhancement-positive mitogen(concanavalin A, ConA) control, and uninfected cell control had anaverage RT titer of 26,518 cpm/mL (1:500 dilution), 54,634 cpm/mL(10,000 dilution), and 2612 cpm/mL (1:500 dilution), respectively(85,000 cpm/mL stock). Four epitope peptides were tested in an in vivoefficacy study with prototype vaccine prime and MAP boosts againstpathogenic FIV challenge (see MAP section). Notably, inhibition of HIV-1infection of normal human PBMC was observed with peptide Fp9-3 (lowestdose, p=0.024) but not with peptide FRT3-4 (FIG. 36B) (92,000 cpm/mLstock).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NOs:1-40 are epitopes contemplated within the scope of theinvention.

SEQ ID NOs:41 and 42 are chimeric polynucleotides of the presentinvention.

SEQ ID NOs:43 and 44 are chimeric polypeptides encoded by a chimericpolynucleotide of the invention.

SEQ ID NOs:45-450 are epitopes contemplated within the scope of theinvention.

SEQ ID NOs: 451-591 are epitopes contemplated within the scope of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention concerns methods and materials for providing animmune response in an animal or person against an immunodeficiencyvirus, such as HIV, SIV, or FIV. In one embodiment, a method of theinvention comprises administering one or more antigens and/or immunogensto the person or animal wherein the antigen or immunogen comprises oneor more epitopes evolutionarily conserved between differentimmunodeficiency viruses. In one embodiment, the epitope is one that isconserved between HIV and SIV, or between HIV and FIV. In anotherembodiment, the epitope is conserved between FIV and SIV. In anotherembodiment, the epitope is one that is conserved between HIV, SIV, andFIV. In one embodiment, where a human is administered the antigen and/orimmunogen, the antigen or immunogen is from an FIV or HIV, and theepitope is evolutionarily conserved between HIV and FIV. In oneembodiment, where the animal is a feline animal, the antigen and/orimmunogen is from an HIV or FIV, and the epitope is evolutionarilyconserved between HIV and FIV. In one embodiment of a method of thepresent invention, the epitope is a T-cell epitope. In a specificembodiment, the epitope induces one or more T cell responses, such asrelease of cytotoxins (e.g., perforin, granzymes, and/or granulysin)and/or cytokines (IFNγ, TNF-α, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13,etc.). In a specific embodiment, the T-cell epitope is a cytotoxic Tlymphocyte (CTL), polyfunctional T cell epitope, and/or T-helper (Th)epitope. Antigens and immunogens of the invention can be peptides and/orproteins that comprise one or more evolutionarily conserved epitopes ofthe invention.

Examples of epitopes contemplated within the scope of the inventioninclude peptides or proteins comprising the amino acid sequence shown inany of SEQ ID NOs:1-40 or in any of SEQ ID NOs:45-591, independently orany possible combination thereof, or in any of the examples, figures ortables of the subject application, or an immunogenic fragment or variantof the amino acid sequence. In a specific embodiment, a peptide orprotein of the invention comprises the amino acid sequence shown in anyof SEQ ID NOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163, 164, 165, 166,167, 176, 177, 178, 179, 214, 215, 216, 217, 218, 288, 301, 303, 304,359, 361, 431, 432, 438, 442, 443, 453, 459, 460, 466, 479, 488, 492,and/or 493. In one embodiment, a plurality of peptides and/or proteinscomprising an epitope of the invention are administered to the person oranimal. For example, in one embodiment, two or more peptides or proteinscomprising the amino acid sequence of any of SEQ ID NOs:10, 21, 22, 23,61, 62, 63, 64, 65, 163, 164, 165, 166, 167, 176, 177, 178, 179, 214,215, 216, 217, 218, 288, 301, 303, 304, 359, 361, 431, 432, 438, 442,443, 453, 459, 460, 466, 479, 488, 492, and/or 493 are administered. Forexample, a first peptide comprising SEQ ID NO:61 and a second peptidecomprising SEQ ID NO:63 can be administered. In another embodiment, apeptide or protein comprising two or more epitopes of the presentinvention is administered to the person or animal. In one embodiment,the peptide or protein can comprise two or more epitopes by linking twoor more peptide sequences of the invention together, or by having apolynucleotide encode two or more peptide sequences together in a singleprotein, and expressing the polynucleotide to produce the protein. Inone embodiment, a peptide or protein comprising two or more amino acidsequences shown in any of SEQ ID NOs:10, 21, 22, 23, 61, 62, 63, 64, 65,163, 164, 165, 166, 167, 176, 177, 178, 179, 214, 215, 216, 217, 218,288, 301, 303, 304, 359, 361, 431, 432, 438, 442, 443, 453, 459, 460,466, 479, 488, 492, and/or 493 is administered to the person or animal.In a specific embodiment, a peptide or protein comprising the amino acidsequence of SEQ ID NO:63 and/or SEQ ID NO:64 is administered to theperson or animal. In yet another embodiment, a peptide or proteinutilized in the present invention comprises an amino acid sequence shownin any of SEQ ID NO:10, SEQ ID NO: 21, SEQ ID NO:22, or SEQ ID NO:23. Ina further embodiment, a peptide or protein utilized in the presentinvention comprises an amino acid sequence shown in any of SEQ IDNOs:176, 177, 178, 179, 214, 215, 216, 217, or 218. In yet a furtherembodiment, a peptide or protein utilized in the present inventioncomprises an amino acid sequence shown in any of SEQ ID NO:288, SEQ IDNO:301, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:359, SEQ ID NO:361, SEQID NO:453, SEQ ID NO:459, SEQ ID NO:460, SEQ ID NO:466, SEQ ID NO:479,SEQ ID NO:488, 492, and/or 493.

In one embodiment, the immune response induced by a method of thepresent invention is a T cell response, such as a CTL-associated immuneresponse and/or a T helper cell response. In a specific embodiment, theimmune response induced by a method of the present invention comprisesCD4+ and/or CD8+ T cell responses, and/or gamma interferon (IFNγ)production. In one embodiment, cytotoxins (such as perforin, granzyme A,granzyme B, etc.) and/or cytokines (IFNγ, IL-4, IL-5, IL-9, IL-10,IL-13, etc.) are produced. In one embodiment, the immune response is aprotective immune response that provides protection to the person oranimal from infection by an immunodeficiency virus. In a specificembodiment, the immune response provides the person or animal withprotection from infection by HIV or FIV. In one embodiment, the personor animal receiving the antigen or immunogen is already infected with animmunodeficiency virus. In another embodiment, the person or animal isnot infected with an immunodeficiency virus prior to administration ofthe antigen or immunogen.

The subject invention also concerns evolutionarily conserved epitopes ofimmunodeficiency viruses. In one embodiment, the epitope is one that isconserved between HIV and SIV, or between HIV and FIV. In anotherembodiment, the epitope is one that is conserved between HIV, SIV, andFIV. In one embodiment, the epitope is a T-cell epitope. In a specificembodiment, the T-cell epitope is a cytotoxic T lymphocyte (CTL)epitope, polyfunctional T cell (CD3+CD4+ and CD3+CD8+ T cells thatexpress multiple cytokines, cytotoxins, chemokines, and functionalactivities such as proliferation) epitope, and/or T-helper (Th) epitope.In one embodiment, the epitopes are from a viral integrase protein. Inanother embodiment, the epitopes are from a viral reverse transcriptase(RT) protein. In a further embodiment, the epitopes are from a viralcore or capsid (p24) protein. Antigens and immunogens of the inventioncan be peptides and/or proteins that comprise one or more evolutionarilyconserved epitopes of the invention. Examples of epitopes contemplatedwithin the scope of the invention include peptides or proteinscomprising the amino acid sequence shown in SEQ ID NOs:1-40 or in any ofSEQ ID NOs:45-591, independently or any possible combination thereof, orin any of the examples, figures or tables of the subject application, oran immunogenic fragment or variant of the amino acid sequence. In aspecific embodiment, an epitope of the invention comprises a peptide orprotein comprising the amino acid sequence shown in any of SEQ IDNOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163, 164, 165, 166, 167, 176,177, 178, 179, 214, 215, 216, 217, 218, 288, 301, 303, 304, 359, 361,431, 432, 438, 442, 443, 453, 459, 460, 466, 479, 488, 492, and/or 493.In another embodiment, an epitope of the invention comprises a peptideor protein comprising two or more amino acid sequences of any of SEQ IDNOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163, 164, 165, 166, 167, 176,177, 178, 179, 214, 215, 216, 217, 218, 288, 301, 303, 304, 359, 361,431, 432, 438, 442, 443, 453, 459, 460, 466, 479, 488, 492, and/or 493.In a specific embodiment, an epitope of the invention comprises apeptide or protein comprising the amino acid sequence of SEQ ID NO:63and/or SEQ ID NO:64. In yet another embodiment, an epitope of theinvention comprises a peptide or protein comprising an amino acidsequence shown in any of SEQ ID NO:10, SEQ ID NO: 21, SEQ ID NO:22, orSEQ ID NO:23. In a further embodiment, an epitope of the inventioncomprises a peptide or protein comprising an amino acid sequence shownin any of SEQ ID NOs:176, 177, 178, 179, 214, 215, 216, 217, or 218. Inyet a further embodiment, an epitope of the invention comprises apeptide or protein comprising an amino acid sequence shown in any of SEQID NO:288, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:304, SEQ ID NO:359,SEQ ID NO:361, SEQ ID NO:453, SEQ ID NO:459, SEQ ID NO:460, SEQ IDNO:466, SEQ ID NO:479, SEQ ID NO:488, SEQ ID NO:492, and/or SEQ IDNO:493. The subject invention also concerns polynucleotides encoding theamino acid sequence of epitopes of the invention.

The subject invention also concerns vaccines comprising one or moreantigens and/or immunogens that comprise or encode evolutionarilyconserved epitopes of the present invention. The vaccine or immunogeniccompositions of the subject invention also encompass recombinant viralvector-based or polynucleotide constructs that may comprise a nucleicacid encoding a peptide or protein comprising an evolutionarilyconserved epitope of the present invention or encoding a chimericpolypeptide of the present invention. Examples of epitopes contemplatedwithin the scope of the invention include peptides or proteinscomprising the amino acid sequence shown in SEQ ID NOs:1-40 or in any ofSEQ ID NOs:45-591, independently or any possible combination thereof, orin any of the examples, figures or tables of the subject application, oran immunogenic fragment or variant of the amino acid sequence. In aspecific embodiment, a peptide or protein of the invention comprises theamino acid sequence shown in any of SEQ ID NOs:10, 21, 22, 23, 61, 62,63, 64, 65, 163, 164, 165, 166, 167, 176, 177, 178, 179, 214, 215, 216,217, 218, 288, 301, 303, 304, 359, 361, 431, 432, 438, 442, 443, 453,459, 460, 466, 479, 488, 492, and/or 493. In another embodiment, apeptide or protein of the invention can comprise two or more amino acidsequences of any of SEQ ID NOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163,164, 165, 166, 167, 176, 177, 178, 179, 214, 215, 216, 217, 218, 288,301, 303, 304, 359, 361, 431, 432, 438, 442, 443, 453, 459, 460, 466,479, 488, 492, and/or 493. In a specific embodiment, a peptide orprotein comprises the amino acid sequence of SEQ ID NO:63_(—) and/or SEQID NO:64. In yet another embodiment, a peptide or protein of the presentinvention comprises an amino acid sequence shown in any of SEQ ID NO:10,SEQ ID NO: 21, SEQ ID NO:22, or SEQ ID NO:23. In a further embodiment, apeptide or protein of the present invention comprises an amino acidsequence shown in any of SEQ ID NOs:176, 177, 178, 179, 214, 215, 216,217, or 218. In yet a further embodiment, a peptide or protein utilizedin the present invention comprises an amino acid sequence shown in anyof SEQ ID NO:288, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:304, SEQ IDNO:359, SEQ ID NO:361, SEQ ID NO:453, SEQ ID NO:459, SEQ ID NO:460, SEQID NO:466, SEQ ID NO:479, SEQ ID NO:488 SEQ ID NO:492, and/or SEQ IDNO:493. In an exemplified embodiment, a chimera polynucleotide comprisesthe sequence shown in SEQ ID NO:41 or SEQ ID NO:42. In a furtherexemplified embodiment, a chimera polypeptide comprises the sequenceshown in SEQ ID NO:43 or SEQ ID NO:44. Any suitable viral vector thatcan be used to prepare a recombinant vector/virus construct iscontemplated for use with the subject invention. For example, viralvectors derived from adenovirus, avipox, herpesvirus, vaccinia,canarypox, entomopox, swinepox, West Nile virus and others known in theart can be used with the compositions and methods of the presentinvention. Recombinant polynucleotide vectors that encode and expresscomponents can be constructed using standard genetic engineeringtechniques known in the art. In addition, the various vaccinecompositions described herein can be used separately and in combinationwith each other. For example, primary immunizations of an animal may userecombinant vector-based constructs, having single or multiple straincomponents, followed by secondary boosts with vaccine compositionscomprising inactivated virus or inactivated virus-infected cell lines.Other immunization protocols with the vaccine compositions of theinvention are apparent to persons skilled in the art and arecontemplated within the scope of the present invention.

The subject invention also concerns compositions comprising epitopesand/or chimeric polypeptides of the invention, or polynucleotidesencoding them. In one embodiment, a composition of the inventioncomprises a pharmaceutically or biologically acceptable carrier,diluent, and/or adjuvant.

The subject invention also concerns antibodies, or an antigen bindingfragment thereof, that bind to HIV, SIV, and/or FIV epitopes. In oneembodiment, an antibody of the invention is a monoclonal antibody. Inone embodiment, an antibody of the invention binds specifically to anHIV protein, e.g., an HIV p24 protein. In a specific embodiment, anantibody of the invention is the monoclonal antibody designated asHL2309 (produced by clone 2B3-1F6) or HL2310 (produced by clone2B3-2A4). In another embodiment, an antibody of the invention bindsspecifically to an FIV protein, e.g., an HIV p24 protein. In a specificembodiment, an antibody of the invention is the monoclonal antibodydesignated as HL2350 (produced by clone 8B2-1E1) or HL2351 (produced byclone 8B2-2A1). In a further embodiment, an antibody of the inventionbinds specifically to both an HIV and an FIV protein, i.e., the antibodycross-reacts with an epitope that is present on both an HIV and an FIVprotein, such as a p24 protein. The subject invention also concerns theepitopes recognized by an antibody of the invention. Table 1 showsmonoclonal antibodies of the present invention and their reactivity withHIV p24 and FIV p24.

TABLE 1 Monoclo- clone isotype/ cross-reac- nal ID number antigen lightchain tivity** HL 2309 2B3-1F6 HIV-1 UCD-1 p24 IgG1/kappa NO*** HL 23102B3-2A4 HIV-1 UCD-1 p24 IgG1/kappa NO*** HL 2311 2B4-1B6 HIV-1 UCD-1 p24IgG1/kappa NO*** HL 2312 2B4-1E8 HIV-1 UCD-1 p24 IgG1/kappa NO*** HL2335 4C3 HIV-1 UCD-1 p24 IgG2b/kappa NO*** HL 2336 5G2 HIV-1 UCD-1 p24IgM/kappa YES HL 2322 9D6 FIV Petaluma p24 IgG1/kappa YES HL 2323 7A3FIV Petaluma p24 IgG1/kappa NO*** HL 2324 2G12 FIV Petaluma p24IgG1/kappa YES HL 2348 9A12-2A3 FIV Petaluma p24 IgG1/kappa YES HL 23499A12-2C2 FIV Petaluma p24 IgG1/kappa YES HL 2350 8B2-1E1 FIV Petalumap24 IgG1/kappa NO*** HL 2351 8B2-2A1 FIV Petaluma p24 IgG1/kappa NO*** *All mouse monoclonal antibodies (MAbs) to HIV-1 p24 are positive byELISA and Weternblot to HIV-1 p24. Similarly, all MAbs to FIV p24 arepositive by ELISA and WB to FIV p24. **Cross-reactivity denotesreactivity of anti-HIV-1 p24 MAbs to FIV p24 and vice versa. ***TheseMAbs differentiate between HIV- p24 and FIV p24 when used in rightcombinations.

The subject invention also concerns expression constructs comprising oneor more polynucleotides of the invention. Expression constructs of theinvention will also generally include regulatory elements that arefunctional in the intended host cell in which the expression constructis to be expressed. Thus, a person of ordinary skill in the art canselect regulatory elements for use in, for example, bacterial hostcells, yeast host cells, plant host cells, insect host cells, mammalianhost cells, and human host cells. Regulatory elements include promoters,transcription termination sequences, translation termination sequences,enhancers, and polyadenylation elements. As used herein, the term“expression construct” refers to a combination of nucleic acid sequencesthat provides for transcription of an operably linked nucleic acidsequence. As used herein, the term “operably linked” refers to ajuxtaposition of the components described wherein the components are ina relationship that permits them to function in their intended manner.In general, operably linked components are in contiguous relation.

An expression construct of the invention can comprise a promotersequence operably linked to a polynucleotide sequence encoding a peptideof the invention. Promoters can be incorporated into a polynucleotideusing standard techniques known in the art. Multiple copies of promotersor multiple promoters can be used in an expression construct of theinvention. In a preferred embodiment, a promoter can be positioned aboutthe same distance from the transcription start site as it is from thetranscription start site in its natural genetic environment. Somevariation in this distance is permitted without substantial decrease inpromoter activity. A transcription start site is typically included inthe expression construct.

For expression in animal cells, an expression construct of the inventioncan comprise suitable promoters that can drive transcription of thepolynucleotide sequence. If the cells are mammalian cells, thenpromoters such as, for example, actin promoter, metallothioneinpromoter, NF-kappaB promoter, EGR promoter, SRE promoter, IL-2 promoter,NFAT promoter, osteocalcin promoter, SV40 early promoter and SV40 latepromoter, Lck promoter, BMP5 promoter, TRP-1 promoter, murine mammarytumor virus long terminal repeat promoter, STAT promoter, or animmunoglobulin promoter can be used in the expression construct.

Expression constructs of the invention may optionally contain atranscription termination sequence, a translation termination sequence,signal peptide sequence, and/or enhancer elements. Transcriptiontermination regions can typically be obtained from the 3′ untranslatedregion of a eukaryotic or viral gene sequence. Transcription terminationsequences can be positioned downstream of a coding sequence to providefor efficient termination. Signal peptides are a group of short aminoterminal sequences that encode information responsible for therelocation of an operably linked peptide to a wide range ofpost-translational cellular destinations, ranging from a specificorganelle compartment to sites of protein action and the extracellularenvironment. Targeting a peptide to an intended cellular and/orextracellular destination through the use of operably linked signalpeptide sequence is contemplated for use with the immunogens of theinvention. Chemical enhancers are cis-acting elements that increase genetranscription and can also be included in the expression construct.Chemical enhancer elements are known in the art, and include, but arenot limited to, the cytomegalovirus (CMV) early promoter enhancerelement and the SV40 enhancer element. DNA sequences which directpolyadenylation of the mRNA encoded by the structural gene can also beincluded in the expression construct.

Unique restriction enzyme sites can be included at the 5′ and 3′ ends ofthe expression construct to allow for insertion into a polynucleotidevector. As used herein, the term “vector” refers to any genetic element,including for example, plasmids, cosmids, chromosomes, phage, virus, andthe like, which is capable of replication when associated with propercontrol elements and which can transfer polynucleotide sequences betweencells. Vectors contain a nucleotide sequence that permits the vector toreplicate in a selected host cell. A number of vectors are available forexpression and/or cloning, and include, but are not limited to, pBR322,pUC series, M13 series, and pBLUESCRIPT vectors (Stratagene, La Jolla,Calif.).

Polynucleotides, vectors, and expression constructs of the invention canbe introduced in vivo via lipofection (DNA transfection via liposomesprepared from synthetic cationic lipids) (Felgner et al., 1987).Synthetic cationic lipids (LIPOFECTIN, Invitrogen Corp., La Jolla,Calif.) can be used to prepare liposomes to encapsulate apolynucleotide, vector, or expression construct of the invention. Apolynucleotide, vector, or expression construct of the invention canalso be introduced as naked DNA using methods known in the art, such astransfection, microinjection, electroporation, calcium phosphateprecipitation, and by biolistic methods.

As used herein, the terms “nucleic acid” and “polynucleotide sequence”refer to a deoxyribonucleotide or ribonucleotide polymer in eithersingle- or double-stranded form, and unless otherwise limited, wouldencompass known analogs of natural nucleotides that can function in asimilar manner as naturally-occurring nucleotides. The polynucleotidesequences include both the DNA strand sequence that is transcribed intoRNA and the RNA sequence that is translated into protein. Thepolynucleotide sequences include both full-length sequences as well asshorter sequences derived from the full-length sequences. It isunderstood that a particular polynucleotide sequence includes thedegenerate codons of the native sequence or sequences which may beintroduced to provide codon preference in a specific host cell. Thepolynucleotide sequences falling within the scope of the subjectinvention further include sequences which specifically hybridize withthe exemplified sequences. The polynucleotide includes both the senseand antisense strands as either individual strands or in the duplex.

The methods of the present invention contemplate a primary immunizationwith an antigen, immunogen, peptide, polypeptide, polynucleotide, and/orcomposition of the invention. Subsequent or secondary immunizations arealso contemplated within the scope of the subject methods. The antigen,immunogen, peptide, polypeptide, polynucleotide, and/or composition usedfor secondary immunizations can be the same as or vary from that usedfor primary immunization. For example, primary immunizations of ananimal may use recombinant vector-based HIV, FIV, or SIV constructs,having single or multiple strain components, followed by secondaryboosts with compositions comprising HIV-, FIV-, or SIV-infected celllines, or HIV, FIV, or SIV polypeptides, or cell free HIV or SIV virus,also having single or multiple strain components. Primary immunizationscan also use an HIV, FIV, and/or SIV DNA vaccine. In one embodiment, arecombinant vector construct is used for the primary immunization,whereas a protein, or protein plus recombinant vector construct, subunitvaccine composition is used for secondary boosts. Other immunizationprotocols with the vaccine compositions of the invention are apparent topersons skilled in the art and are contemplated within the scope of thepresent invention.

The antibodies can be polyclonal or monoclonal in form. The antibodiescan be derived from any animal capable of producing antibodies to theepitopes, and include, for example, human, ape, monkey, mouse, rat,goat, sheep, pig, cow, and feline animals. Also contemplated within thescope of the invention are non-human antibodies that have been“humanized” using standard procedures known in the art, such as thosedescribed in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762; 6,180,370;and 6,407,213.

An antibody that is contemplated for use in the present invention can bein any of a variety of forms, including a whole immunoglobulin, anantibody fragment such as Fv, Fab, and similar fragments, as well as asingle chain antibody that includes the variable domain complementaritydetermining regions (CDR), and similar forms, all of which fall underthe broad term “antibody,” as used herein.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment of an antibody yields an F(ab′)₂fragment that has two antigen binding fragments, which are capable ofcross-linking antigen, and a residual other fragment (which is termedpFc′). Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “antigen binding fragment” withrespect to antibodies, refers to, for example, Fv, F(ab) and F(ab′)₂fragments.

Antibody fragments can retain an ability to selectively bind with theantigen or analyte are contemplated within the scope of the inventionand include:

(1) Fab is the fragment of an antibody that contains a monovalentantigen-binding fragment of an antibody molecule. A Fab fragment can beproduced by digestion of whole antibody with the enzyme papain to yieldan intact light chain and a portion of one heavy chain.

(2) Fab′ is the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain. Two Fab′ fragmentsare obtained per antibody molecule. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region.

(3) (Fab′)₂ is the fragment of an antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction. F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H)-V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen-bindingsite on the surface of the V_(H)-V_(L) dimer. Collectively, the six CDRsconfer antigen-binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain (V_(L)), thevariable region of the heavy chain (V_(H)), linked by a suitablepolypeptide linker as a genetically fused single chain molecule. Suchsingle chain antibodies are also referred to as “single-chain Fv” or“sFv” antibody fragments. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv fragments, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y.,pp. 269 315 (1994).

Antibodies within the scope of the invention can be of any isotype,including IgG, IgA, IgE, IgD, and IgM. IgG isotype antibodies can befurther subdivided into IgG1, IgG2, IgG3, and IgG4 subtypes. IgAantibodies can be further subdivided into IgA1 and IgA2 subtypes.

Antibodies to be used in the subject invention can be genus or speciesspecific to a target cell. Antibodies of the invention can be preparedusing standard techniques known in the art. Antibodies useful in theinvention can be polyclonal or monoclonal antibodies. Monoclonalantibodies can be prepared using standard methods known in the art(Kohler et al., 1975).

The subject invention also concerns hybridomas that produce monoclonalantibodies of the present invention.

Peptide and/or polypeptide antigens and immunogens of the presentinvention can also be provided in the form of a multiple antigenicpeptide (MAP) construct, with or without lypophylic attachment to eachpeptide string. The preparation of MAP constructs has been described inTam (1988) and Kowalczyk et al. (2010). MAP constructs utilize a corematrix of lysine residues onto which multiple copies of an immunogen aresynthesized (Posnett et al., 1988). In one embodiment, MAP constructs ofthe invention can comprise one or more fatty acids attached to the corematrix. The fatty acid can comprise from about 4 to about 48 or morecarbon atoms, and can be saturated and/or unsaturated. In a specificembodiment, the fatty acid is palmitic acid (hexadecanoic acid).Multiple MAP constructs, each containing the same or differentimmunogens, can be prepared and administered in a vaccine composition inaccordance with methods of the present invention. In one embodiment, thesame or different peptides are linked end to end. The same or differentpeptides can be linked directly to each other (i.e., without a linkersequence) or they can be linked via a linker moiety such as a shortamino acid sequence (e.g., a furin-sensitive linker), examples of whichinclude, but are not limited to, peptides comprising SEQ ID NO:493. Inone embodiment, a MAP construct is provided with and/or administeredwith one or more adjuvants. In one embodiment, a MAP of the inventioncomprises one or more peptides that comprise the amino acid sequences ofone or more of SEQ ID NOs:1-40 or 45-591.

Natural, recombinant or synthetic polypeptides of immunodeficiency viralproteins, and peptide fragments thereof, can also be used as vaccinecompositions according to the subject methods. Procedures for preparingFIV, SIV, and HIV polypeptides are well known in the art. For example,FIV, SIV, and HIV polypeptides can be synthesized using solid-phasesynthesis methods (Merrifield, 1963). FIV, SIV, and HIV polypeptides canalso be produced using recombinant DNA techniques wherein apolynucleotide molecule encoding an FIV, SIV, or HIV protein or peptideis expressed in a host cell, such as bacteria, yeast, or mammalian celllines, and the expressed protein purified using standard techniques ofthe art.

According to the methods of the subject invention, the antigenic andimmunogenic compositions described herein can be administered tosusceptible hosts in an effective amount and manner to induce an immuneresponse and/or protective immunity against subsequent challenge orinfection of the host by FIV, SIV, or HIV. The immunogens are typicallyadministered parenterally, by injection, for example, eithersubcutaneously, intradermally, intraperitoneally, or intramuscularly, orby oral or nasal administration, or any combination of such routes ofadministration. Usually, the immunogens are administered to a hostanimal at least two times, with an interval of one or more weeks betweeneach administration. However, other regimens for the initial and boosteradministrations of the immunogens are contemplated, and may depend onthe judgment of the practitioner and the particular host animal beingtreated.

Antigens and immunogens that can be used in accordance with the presentinvention can be provided with a pharmaceutically-acceptable carrier ordiluent. Compounds and compositions useful in the subject invention canbe formulated according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in detail in a number ofsources which are well known and readily available to those skilled inthe art. For example, Remington's Pharmaceutical Science by E. W.Martin, Easton Pa., Mack Publishing Company, 19^(th) ed., 1995,describes formulations which can be used in connection with the subjectinvention. In general, the compositions of the subject invention will beformulated such that an effective amount of an antigen or immunogen iscombined with a suitable carrier in order to facilitate effectiveadministration of the composition. The compositions used in the presentmethods can also be in a variety of forms. These include, for example,solid, semi-solid, and liquid dosage forms, such as tablets, pills,powders, liquid solutions or suspension, suppositories, injectable andinfusible solutions, and sprays. The preferred form depends on theintended mode of administration and therapeutic application. Thecompositions also preferably include conventional pharmaceuticallyacceptable carriers and diluents which are known to those skilled in theart. Examples of carriers or diluents for use with the subjectpeptidomimetics include, but are not limited to, water, saline, oilsincluding mineral oil, ethanol, dimethyl sulfoxide, gelatin,cyclodextrans, magnesium stearate, dextrose, cellulose, sugars, calciumcarbonate, glycerol, alumina, starch, and equivalent carriers anddiluents, or mixtures of any of these. Formulations of an immunogen ofthe invention can also comprise suspension agents, protectants,lubricants, buffers, preservatives, and stabilizers. To provide for theadministration of such dosages for the desired therapeutic treatment,pharmaceutical compositions of the invention will advantageouslycomprise between about 0.1% and 45%, and especially, 1 and 15% by weightof the antigen, antigens, immunogen or immunogens based on the weight ofthe total composition including carrier or diluent.

The immunogenic compositions of the subject invention can be prepared byprocedures well known in the art. For example, the antigens orimmunogens are typically prepared as injectables, e.g., liquid solutionsor suspensions. The antigens or immunogens are administered in a mannerthat is compatible with dosage formulation, and in such amount as willbe therapeutically effective and immunogenic in the recipient. Theoptimal dosages and administration patterns for a particular antigen orimmunogen formulation can be readily determined by a person skilled inthe art.

Virus and cells in an antigenic or immunogenic formulation may beinactivated or attenuated using methods known in the art. The amount ofcell-free whole or partial virus in a vaccine dose will usually be inthe range from about 0.1 mg to about 5 mg, and more usually being fromabout 0.2 mg to about 2 mg. The dosage for formulations comprisingvirus-infected cell lines will usually contain from about 10⁶ to about10⁸ cells per dose, and more usually from about 5×10⁶ to about 7.5×10⁷cells per dose. The amount of protein or peptide immunogen in a dose fora feline animal can vary from about 0.1 μg to 10000 μg, or about 1 μg to5000 μg, or about 10 μg to 1000 μg, or about 25 μg to 750 μg, or about50 μg to 500 μg, or 100 μg to 250 μg, depending upon the size, age,etc., of the animal receiving the dose.

In one embodiment, an antigen or immunogen of the invention is providedwith one or more adjuvants that increase the person or animal's immuneresponse against the antigen or immunogen. Antigens and immunogens ofthe invention can be provided with and/or administered with any suitableadjuvant or adjuvants known in the art. In one embodiment, the adjuvantis one that helps induce a strong cellular immune response. Adjuvantsthat can be used in the antigen and immunogen formulations of theinvention include threonyl muramyl dipeptide (MDP) (Byars et al., 1987),Ribi adjuvant system components (Corixa Corp., Seattle, Wash.) includingthe cell wall skeleton (CWS) component, Freund's complete, and Freund'sincomplete adjuvants, bacterial lipopolysaccharide (LPS), such as fromE. coli, or a combination thereof. A variety of other adjuvants suitablefor use with the methods and vaccines of the subject invention, such asalum, aluminum hydroxide, and saponin are well known in the art and arecontemplated for use with the subject invention. Cytokines (γ-IFN,GM-CSF, CSF, etc.) and lymphokines and interleukins (IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8. IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, and IL-22) have alsobeen used as adjuvants and/or supplements to vaccine compositions andare contemplated within the scope of the present invention. One or moredifferent cytokines and lymphokines can be included in a compositioncomprising an antigen or immunogen of the invention. In one embodiment,an antigen or immunogen of the invention is administered to an animal incombination with the lymphokine interleukin-12 (IL-12) in combinationwith another adjuvant. Also specifically contemplated within the scopeof the invention is the use of the lymphokine interleukin-18 (IL-18) aspart of an adjuvant composition. In one embodiment, an adjuvantcomposition used with the subject invention comprises a combination ofIL-12 and IL-15, or IL-15 and IL-18, or IL-12 and IL-18, or IL-12,IL-15, and IL-18. The cytokine selected is of a species that hasbiological activity in the animal receiving the antigen or immunogen.For example, if the animal is a cat, then the cytokine can be a humancytokine or a feline cytokine, e.g., feline IL-12, feline IL-15, felineIL-18, etc.

Abbreviations of FIV strains used herein are shown below:

Strain (subtype) Abbreviation Petaluma (A) FIV_(Pet) Dixon (A) FIV_(Dix)UK8 (A) FIV_(UK8) Bangston (B) FIV_(Bang) Aomori-1 (B) FIV_(Aom1)Aomori-2 (B) FIV_(Aom2) FC1 (B) FIV_(FC1) Shizuoka (D) FIV_(Shi)Dutch113 (A) FIV_(Dut113) Dutch19K (A) FIV_(Dut19) UK2 (A) FIV_(UK2)SwissZ2 (A) FIV_(SwiZ2) Sendai-1 (A) FIV_(Sen1) Sendai-2 (B) FIV_(Sen2)USCAzepy01A (A) FIV USCAhnky11A (A) FIV_(USC11) USCAtt-10A (A)FIV_(USC10) USCAlemy01 (A) FIV USCAsam-01A (A) FIV PPR (A) FIV_(PPR)FranceWo FIV_(Fra) Netherlands FIV_(Net) USILbrny03B (B) FIV_(USI03) TM2(B) FIV_(TM2) USCKlgri02B (B) FIV_(USC02) Yokohama (B) FIV_(Yok)USMAsboy03B (B) FIV_(USMA03) USTXmtex03B (B) FIV_(UST03) USMCglwd03B (B)FIV_(USMC03) CABCpbar03C (C) FIV_(CAB03) CABCpbar07C (C) FIV_(CAB07)CABCpady02C (C) FIV_(CAB02) Fukuoka (D) FIV_(Fuku)

Antigens and immunogens of the invention are typically administeredparenterally, by injection, for example, either subcutaneously,intradermally, intraperitoneally, or intramuscularly. Other suitablemodes of administration include oral or nasal administration. Usually,the antigens and immunogens are administered to a human or animal atleast two times, with an interval of one or more weeks between eachadministration. However, other regimens for the initial and boosteradministrations of the antigens and immunogens are contemplated, and maydepend on the judgment of the practitioner and the patient beingtreated.

Antigenic and immunogenic compositions of the subject invention can beprepared by procedures well known in the art. For example, the antigensand immunogens are typically prepared as injectables, e.g., liquidsolutions or suspensions. The antigens and immunogens are administeredin a manner that is compatible with dosage formulation, and in suchamount as will be therapeutically effective and immunogenic in therecipient. The optimal dosages and administration patterns for aparticular antigen and immunogen formulation can be readily determinedby a person skilled in the art.

Antigens and immunogens that can be used in accordance with the presentinvention can be provided with a pharmaceutically-acceptable carrier ordiluent. In one embodiment, an antigen or immunogen of the invention isprovided with one or more adjuvants that increase the human or animal'simmune response against the antigen or immunogen. Antigens andimmunogens of the invention can be provided with and/or administeredwith any suitable adjuvant or adjuvants known in the art.

The antigenic or immunogenic peptides contemplated in the subjectinvention include the specific peptides exemplified herein as well asequivalent peptides which may be, for example, somewhat longer orshorter than the peptides exemplified herein. For example, using theteachings provided herein, a person skilled in the art could readilymake peptides having from 1 to about 15 or more amino acids added to, or1 to 10 amino acids removed from, either or both ends of the disclosedpeptides using standard techniques known in the art. Any added aminoacids can be different or the same as the corresponding amino acids ofthe full-length protein from which the peptide is derived. The skilledartisan, having the benefit of the teachings disclosed in the subjectapplication, could easily determine whether a longer or shorter peptideretained the immunogenic activity of the specific peptides exemplifiedherein.

Substitution of amino acids other than those specifically exemplified ornaturally present in a peptide of the invention are also contemplatedwithin the scope of the present invention. For example, non-naturalamino acids can be substituted for the amino acids of a peptide, so longas the peptide having the substituted amino acids retains substantiallythe same immunogenic activity as the peptide in which amino acids havenot been substituted. Examples of non-natural amino acids include, butare not limited to, ornithine, citrulline, hydroxyproline, homoserine,phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γ-aminobutyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-aminoisobutyric acid, 3-amino propionic acid, norleucine, norvaline,sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, C-methyl amino acids,N-methyl amino acids, and amino acid analogues in general. Non-naturalamino acids also include amino acids having derivatized side groups.Furthermore, any of the amino acids in the protein can be of the D(dextrorotary) form or L (levorotary) form.

Amino acids can be generally categorized in the following classes:non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby a peptide of the present invention having an aminoacid of one class is replaced with another amino acid of the same classfall within the scope of the subject invention so long as the peptidehaving the substitution still retains substantially the same immunogenicactivity as the peptide that does not have the substitution. Table 2below provides a listing of examples of amino acids belonging to eachclass.

TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

Polynucleotides encoding a specifically exemplified peptide or chimericpolypeptide of the invention, or a shorter or longer peptide or chimericpolypeptide, or a peptide having one or more amino acid substitutions inthe sequence are contemplated within the scope of the present invention.The subject invention also concerns variants of the polynucleotides ofthe present invention that encode a peptide of the invention. Variantsequences include those sequences wherein one or more nucleotides of thesequence have been substituted, deleted, and/or inserted. Thenucleotides that can be substituted for natural nucleotides of DNA havea base moiety that can include, but is not limited to, inosine,5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine,5-methylcytosine, and tritylated bases. The sugar moiety of thenucleotide in a sequence can also be modified and includes, but is notlimited to, arabinose, xylulose, and hexose. In addition, the adenine,cytosine, guanine, thymine, and uracil bases of the nucleotides can bemodified with acetyl, methyl, and/or thio groups. Sequences containingnucleotide substitutions, deletions, and/or insertions can be preparedand tested using standard techniques known in the art.

Fragments and variants of a peptide or a chimeric polypeptide of thepresent invention can be generated as described herein and tested forthe presence of immunogenic activity using standard techniques known inthe art.

Polynucleotides, peptides, and chimeric polypeptides contemplated withinthe scope of the subject invention can also be defined in terms of moreparticular identity and/or similarity ranges with those sequences of theinvention specifically exemplified herein. The sequence identity willtypically be greater than 60%, preferably greater than 75%, morepreferably greater than 80%, even more preferably greater than 90%, andcan be greater than 95%. The identity and/or similarity of a sequencecan be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%as compared to a sequence exemplified herein. Unless otherwisespecified, as used herein percent sequence identity and/or similarity oftwo sequences can be determined using the algorithm of Karlin andAltschul (1990), modified as in Karlin and Altschul (1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (1990). BLAST searches can be performed with the NBLASTprogram, score=100, wordlength=12, to obtain sequences with the desiredpercent sequence identity. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be used as described in Altschul et al.(1997). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (NBLAST and XBLAST) can be used.See Worldwide Website: ncbi.nlm.nih.gov.

Factors affecting the preferred dosage regimen may include, for example,the age, weight, sex, diet, activity, lung size, and condition of thesubject; the route of administration; the efficacy, safety, andduration-of-immunity profiles of the particular vaccine used; whether adelivery system is used; and whether the vaccine is administered as partof a drug and/or vaccine combination. Thus, the dosage actually employedcan vary for specific animals, and, therefore, can deviate from thetypical dosages set forth above. Determining such dosage adjustments isgenerally within the skill of those in the art using conventional means.It should further be noted that live attenuated viruses are generallyself-propagating; thus, the specific amount of such a virus administeredis not necessarily critical.

It is contemplated that the vaccine may be administered to the patient asingle time; or, alternatively, two or more times over days, weeks,months, or years. In some embodiments, the vaccine is administered atleast two times. In some such embodiments, for example, the vaccine isadministered twice, with the second dose (e.g., the booster) beingadministered at least about 2 weeks after the first. In someembodiments, the vaccine is administered twice, with the second dosebeing administered no greater than 8 weeks after the first. In someembodiments, the second dose is administered at from about 2 weeks toabout 4 years after the first dose, from about 2 to about 8 weeks afterthe first dose, or from about 3 to about 4 weeks after the first dose.In some embodiments, the second dose is administered about 4 weeks afterthe first dose. In the above embodiments, the first and subsequentdosages may vary, such as, for example, in amount and/or form. Often,however, the dosages are the same as to amount and form. When only asingle dose is administered, the amount of vaccine in that dose alonegenerally comprises a therapeutically effective amount of the vaccine.When, however, more than one dose is administered, the amounts ofvaccine in those doses together may constitute a therapeuticallyeffective amount.

In some embodiments, the vaccine is administered before the recipient isinfected with virus. In such embodiments, the vaccine may, for example,be administered to prevent, reduce the risk of, or delay the onset ofone or more (typically two or more) clinical symptoms.

In some embodiments, the vaccine is administered after the recipient isinfected with influenza. In such embodiments, the vaccine may, forexample, ameliorate, suppress, or eradicate the virus or one or more(typically two or more) clinical symptoms.

It is contemplated that the vaccine may be administered via the felinepatient's drinking water and/or food. It is further contemplated thatthe vaccine may be administered in the form of a treat or toy.

“Parenteral administration” includes subcutaneous injections, submucosalinjections, intravenous injections, intramuscular injections,intrasternal injections, transcutaneous injections, and infusion.Injectable preparations (e.g., sterile injectable aqueous or oleaginoussuspensions) can be formulated according to the known art using suitableexcipients, such as vehicles, solvents, dispersing, wetting agents,emulsifying agents, and/or suspending agents. These typically include,for example, water, saline, dextrose, glycerol, ethanol, corn oil,cottonseed oil, peanut oil, sesame oil, benzyl alcohol, benzyl alcohol,1,3-butanediol, Ringer's solution, isotonic sodium chloride solution,bland fixed oils (e.g., synthetic mono- or diglycerides), fatty acids(e.g., oleic acid), dimethyl acetamide, surfactants (e.g., ionic andnon-ionic detergents), propylene glycol, and/or polyethylene glycols.Excipients also may include small amounts of other auxiliary substances,such as pH buffering agents.

The vaccine may include one or more excipients that enhance a patient'simmune response (which may include an antibody response, cellularresponse, or both), thereby increasing the effectiveness of the vaccine.Use of such excipients (or “adjuvants”) may be particularly beneficialwhen using an inactivated vaccine. The adjuvant(s) may be a substancethat has a direct (e.g., cytokine or Bacillé Calmette-Guerin (“BCG”)) orindirect effect (liposomes) on cells of the patient's immune system.Examples of often suitable adjuvants include oils (e.g., mineral oils),metallic salts (e.g., aluminum hydroxide or aluminum phosphate),bacterial components (e.g., bacterial liposaccharides, Freund'sadjuvants, and/or MDP), plant components (e.g., Quil A), and/or one ormore substances that have a carrier effect (e.g., bentonite, latexparticles, liposomes, and/or Quil A, ISCOM). It should be recognizedthat this invention encompasses both vaccines that comprise anadjuvant(s), as well as vaccines that do not comprise any adjuvant.

It is contemplated that the vaccine may be freeze-dried (or otherwisereduced in liquid volume) for storage, and then reconstituted in aliquid before or at the time of administration. Such reconstitution maybe achieved using, for example, vaccine-grade water.

The present invention further comprises kits that are suitable for usein performing the methods described above. The kit comprises a dosageform comprising a vaccine described above. The kit also comprises atleast one additional component, and, typically, instructions for usingthe vaccine with the additional component(s). The additionalcomponent(s) may, for example, be one or more additional ingredients(such as, for example, one or more of the excipients discussed above,food, and/or a treat) that can be mixed with the vaccine before orduring administration. The additional component(s) may alternatively (oradditionally) comprise one or more apparatuses for administering thevaccine to the patient. Such an apparatus may be, for example, asyringe, inhaler, nebulizer, pipette, forceps, or any medicallyacceptable delivery vehicle. In some embodiments, the apparatus issuitable for subcutaneous administration of the vaccine. In someembodiments, the apparatus is suitable for intranasal administration ofthe vaccine.

Other excipients and modes of administration known in the pharmaceuticalor biologics arts also may be used.

The subject invention also concerns a method for selecting antigensand/or immunogens for use in a vaccine against an immunodeficiencyvirus, such as HIV or FIV, wherein the method comprises identifyingevolutionarily conserved epitopes of the target protein from two or moreimmunodeficiency viruses, wherein one or more of the identified epitopesare selected for use as an antigen or immunogen in the vaccine. In oneembodiment, one or more overlapping peptides of an FIV protein and acorresponding HIV protein, or an FIV protein and a corresponding SIVprotein, or an SIV protein and a corresponding HIV protein are assayedto identify those that are capable of inducing one or more T cellresponses (cell mediated immune responses). Cells are contacted with theone or more peptides for a period of time and then assays are conductedto determine if one or more T cell responses was induced. In oneembodiment, a response assayed for is IFNγ production. In anotherembodiment, a response assayed for is induction of T cell proliferation,such as proliferation of CD4⁺ and/or CD8⁺ T cells. In anotherembodiment, a response assayed for is the production and/or expressionof cytotoxic T cell-associated molecules (e.g., cytotoxins), such asgranzyme A, granzyme B, perforin, and/or CD107a. In one embodiment, amethod of the invention comprises testing one or more peptides forinduction of IFNγ production by cells (e.g., peripheral bloodmononuclear cells (PBMS)) using an enzyme-linked immunosorbent spot(ELISpot) assay for IFNγ. In one embodiment, a method of the inventioncomprises testing one or more peptides for induction of T cellproliferation using a carboxyfluorescein diacetate succinimide ester(CFSE) proliferation assay. The assays contemplated for determining theinduction of a T cell response can provide quantitative and/orqualitative results. In one embodiment, the cells contacted with the oneor more peptides are cells from a feline animal. In one embodiment, thefeline animal is infected with FIV or has been vaccinated against FIV.In another embodiment, the feline animal has not been infected with FIVor vaccinated against FIV. In another embodiment, the cells are from aprimate or a human. In one embodiment, the primate or human has not beeninfected with HIV. In another embodiment, the primate or human has beeninfected with HIV (HIV+). In one embodiment, the HIV+ subject is along-term survivor (LTS). In another embodiment, the subject is ashort-term (ST) survivor. The HIV+ subject can be one that has receivedantiretroviral therapy (ART) or one that has not received ART.

The subject invention also concerns chimeric polynucleotides andpolypeptides that comprise sequences from more than one immunodeficiencyvirus. In one embodiment, a chimera of the invention comprises sequencesof HIV and FIV. In a specific embodiment, a chimera of the invention isa chimeric Gag protein wherein matrix (MA) and nucleocapsid (NC)sequences are from FIV and wherein the core or capsid (CA) (p24)sequences are from an HIV. In an exemplified embodiment, a chimerapolynucleotide comprises the sequence shown in SEQ ID NO:41 or SEQ IDNO:42. In a further exemplified embodiment, a chimera polypeptidecomprises the sequence shown in SEQ ID NO:43 or SEQ ID NO:44. Thesubject invention contemplates that HIV proteins can be substituted forcorresponding FIV proteins in other chimeric polynucleotides andpolypeptides of the invention. For example, HIV pol sequences can besubstituted into corresponding FIV pol sequences.

The subject invention also concerns methods for determining whether ananimal, such as a feline animal, has been vaccinated against FIV with anFIV vaccine of the present invention, or is infected by FIV or has beeninfected by FIV. In one embodiment, a biological sample, such as a bloodor serum sample, is obtained from a feline animal, and the sample isassayed to determine whether the animal has antibodies that bindspecifically to HIV antigens. In a specific embodiment, if an animal isvaccinated with a chimeric polynucleotide or polypeptide of the presentinvention wherein p24 of FIV is replaced with p24 of HIV, thenantibodies specific for the HIV p24 will be present in the animal andcan be detected. In one embodiment, a chimera polypeptide comprises thesequence shown in SEQ ID NO:43 or SEQ ID NO:44. If an animal has beeninfected with FIV, then that animal will not have antibodies that bindto certain HIV p24 epitopes. If an animal has been vaccinated with achimera polypeptide comprising an HIV protein and an FIV protein, thenthe animal will have antibodes that bind to HIV. Epitopes of an HIVprotein that are only recognized by HIV antibodies and that are notrecognized by FIV antibodies can be used in the subject invention.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Selection of HIV Immunogens and Conserved Epitopes

Careful design of vaccine immunogens for protection against a widenumber of HIV variants will be required to deal with the large antigenicdiversity. Conserved viral antigens, subtype-matched antigens, consensusantigens, variants of single antigens and multiple antigens have allbeen used alone or in combination (Li et al. (2007); Korber et al.(2009)). Table 7 shows a few examples for each of the strategies. CTLresponses have been shown to preferentially target the conserved regionsover the more variable ones (Cao et al. (1997)), and these responseshave been associated with better HIV disease outcomes or no diseasemanifestation (Kiepiela et al. (2007); Rowland-Jones et al. (1998);Johnson et al. (1991)).

The most conserved regions of HIV, especially those conserved acrosssubtypes (Korber et al. (2009)) or among lentiviruses (Yamamoto et al.(2010)), may be the best targets of the immune system for inducingvaccine protection. Some of these regions may be protective and are lesslikely to mutate because they hold a functional or structural importanceto the virus species (possibly to the genus); a mutation would induceimpairment to viral fitness (Santra et al. (2010); Barouch et al.(2010)). This possibility makes the identification of conserved epitopesan important aspect of immunogen selection in vaccine design. One meansof including these conserved regions is to construct polyvalent mosaicproteins as vaccine immunogens; thus far, preclinical evaluations of themosaic vaccine have demonstrated great potential for broad T-cellresponses, across subtypes (Korber et al. (2009); Smith (2004); Wang etal. (2009)).

A method of selecting highly conserved regions is to identify those withthe lowest entropy, which is the lowest variability at each aa position.Based on this concept, the most conserved HIV proteins have been shownto be (in order of lowest variability): integrase (IN), core capsid(Gag-p24), reverse transcriptase (RT), and protease (PR) (Table 3)(Yusim et al. (2002)). They were followed by Vpr, Vif, matrix (Gag-p17),Nef, Rev, and the surface envelope (SU-Env). Tat and Vpu have thehighest variability (Table 3). This observation suggests that theselection of conserved vaccine epitopes should be done first from IN,Gag-p24, RT, and PR.

While Jenner may not have considered functional conservation whendeveloping his smallpox vaccine, he can be considered to have been thefirst developer of a vaccine that was based on conserved featuresbetween two different viral species (Jenner (1798)). In a similarfashion, comparisons with other lentiviruses could help identify highlyconserved epitopes that are required for viral function and survival.FIV is a lentivirus that is only distantly related to HIV-1, but maystill be relevant to the evolutionary conserved approach of vaccinedevelopment because of the shared similarities between the HIV and FIVviruses in terms of aa sequence, structure, and pathogenesis (Yamamotoet al. (2007)). A comparison of the aa composition of proteins betweenHIV-1 and FIV demonstrates the following percentages ofidentity/homology: RT, 47/72; IN, 37/65; Gag-p24, 32/63; nucleocapsid(Gag-p7), 30/54; PR, 24/48; Gag-p17, 20/50; SU-Env, 19/43; transmembraneenvelope (TM-Env) 18/42 (Yamamoto et al. (2010)) (Table 3). The threemost conserved proteins are also those that have the lowest entropycalculation, as shown in Table 3 (Yusim et al. (2002)). Hence, the IN,RT, and Gag-p24 proteins appear to be excellent targets for identifyingevolutionary conserved regions that may also contain conserved T-cellepitopes.

TABLE 3 HIV-1/FIV proteins. IN Gag-p24 RT PR Gag-p17 SU-Env Ref.Approximate 0.16 0.18 0.21 0.23 0.45 0.6 † average entropy scores ^(a)HIV/FIV 37/65 32/63 47/72 24/48 20/50 19/43 ‡ protein % aa identity/homology ^(b) ^(a) The average Shannon entropy score is the averagevalue of variability of a given protein at each aa position, calculatedby using many aligned sequences. The approximate values shown arederived from the figure of HIV-1 (group M) protein variability fromYusim et al (Yusim et al. (2002))), where the proteins are presentedfrom lowest to highest variability. Lower scores represent lowervariability and therefore higher aa conservation. ^(b)The percentage ofaa identity and homology between HIV and FIV proteins are shown, withthe three most conserved HIV and FIV proteins bolded. † (Yusim et al.(2002)) ‡ (Yamamoto et al. (2010))

Example 2 Identification of Evolutionary Conserved HIV CTL Epitopes: Useof FIV Proteins

Immunoninformatics has become an integral part in the design of newvaccines with great promise of rapid and effective vaccine discovery(Ardito et al. (2011); Moss et al. (2011); De Groot et al. (2008)). Anumber of tools and databases are now available online including HLAclass-I and -II binding predictions (Los Alamos National Laboratory. HIVmolecular immunology database: Best-defined CTL/CD8⁺ Epitope Summary:(www.hiv.lanl.gov/content/immunology/tables/optimal_ctl_(—)summary.html); Yongqun et al. (2010)), and a number of tools that areuseful for the prediction of CTL epitopes (Table 4). In one studyperformed by our group, NetCTL-1.2 was used to identify CTL epitopes onthe integrase sequences of HIV, SIV and FIV (FIG. 1A). For the twelveHLA supertypes shown in FIG. 1, a large number of CTL epitopes werepredicted on each integrase sequence regardless of the virus: HIV with78 epitopes, SIV with 74 epitopes, and FIV with 85 epitopes. Some ofthese were conserved between HIV and SIV (34 epitopes), as well asbetween HIV and FIV (25 epitopes) (FIG. 1B). A smaller number (17epitopes) was conserved among all three viruses, reducing the targetepitopes to the expected most evolutionary conserved.

TABLE 4 CTL-epitope prediction tools. Name Website Developer Ref.CTLPred www.imtech.res.in/raghava/ctlpred/ India † NetCTLwww.cbs.dtu.dk/services/NetCTL/ Denmark ‡ NetCTLpanwww.cbs.dtu.dk/services/NetCTLpan/ Denmark § † Bhasin et al. (2004) ‡Larsen et al. (2007) § Stranzl et al. (2010)

Thirteen HIV CTL epitopes termed best-defined CTL epitopes have beenidentified empirically on HIV integrase by different laboratories andcompiled on the Los Alamos National Laboratory (LANL) website (Table 5).In this regard, based on observations using SIV and FIV, an evolutionaryconserved HIV CTL epitope can be defined as a CTL epitope with a director indirect SIV and/or FIV CTL counterpart (FIG. 2). Using the directcounterpart approach (FIG. 2, arrow a), three of these epitopes arepredicted to be CTL epitopes conserved between HIV, SIV, and FIV and onewas shown to be an indirect FIV counterpart (Table 6). They share thesame HLA binding and CTL supertype predictions (Larsen et al. (2007);Lundegaard et al. (2008)). The evolutionary conserved (FIG. 2, arrow c).An indirect counterpart to an HIV epitope (bolded in Table 6), locatedupstream on FIV integrase, has higher aa identity and homology than thedirect counterpart (FIG. 2, arrow b). This indirect FIV counterpart hasthe same binding alleles and predicted CTL supertype as the HIV epitope.

TABLE 5 Best defined CTL epitopes on HIV integrase^(a). Position onEpitope HXB2 HLA 1 LPPIVAKEI 28-36 B42 (SEQ ID NO: 1) 2 THLEGKIIL 66-74B*1510 (SEQ ID NO: 2) 3 STTVKAACWW 123-132 B57 (SEQ ID NO: 3) 4IQQEFGIPY 135-143 B*1503 (SEQ ID NO: 4) 5 VRDQAEHL 165-172 Cw18 (SEQ IDNO: 5) 6 KTAVQMAVF 173-181 B*5701 (SEQ ID NO: 6) 7 AVFIHNFKRK 179-188A*0301, A*1101 (SEQ ID NO: 7) 8 FKRKGGIGGY 185-194 B*1503 (SEQ ID NO: 8)9 KRKGGIGGY 186-194 B*2705 (SEQ ID NO: 9) 10 IIATDIQTK 203-211 A*1101(SEQ ID NO: 10) 11 KIQNFRVYY 219-227 A*3002 (SEQ ID NO: 11) 12 VPRRKAKII260-268 B42 (SEQ ID NO: 12) 13 RKAKIIRDY 263-271 B*1503 (SEQ ID NO: 13)^(a)Adapted from LANL(hiv.lanl.gov/content/immunology/tables/optimal_ctl_summary.html) whichwas last updated on 2009-08-31. The best defined CTL epitopes or “Alist” represent the epitopes whose specific HLA class I allele has beendemonstrated with strong certainty and are judged to be at their optimallength.

TABLE 6 HIV-1 integrase CTL epitopes and direct FIV counterparts. IEDBPrediction: Supertype Binding (Total # of Allele Allele binding NetCTL(supertype) Virus Epitopes Iden. Hom. (nM value) alleles) SupertypeB*1510 (B39) HIV THLEGKIIL B*3901(9); B39(2) B39 (SEQ ID NO: 2)B*1501(425) SIV THLEGKIII 78 100 B*3901(44) B39(1) B39 (SEQ ID NO: 14)FIV THFNGKIII 56 78 B*3901(64); B39(2) B39 (SEQ ID NO: 15) B*1501(373)A*0301 (A3) HIV MAVFIHNFK A*0301 A3(5); A1(1) A3 A*1101 (A3) (SEQ ID NO:16) (363); A*1101 (20) SIV MAVHCMNFK 67 67 A*0301(174); A3(4); A1(1) A3(SEQ ID NO: 17) A*1101(25) FIV LALYCLNFK 44 78 A*3001(113); A3(3); A1(1)A3 (SEQ ID NO: 18) A*1101(55) B42 (B7) HIV VPRRKAKII B*0702(43); B7(1);B8(1) B7; B8 (SEQ ID NO: 12) B*0801(53) SIV VPRRKAKII 100 100B*0702(43); B7(1); B8(1) B7; B8 (SEQ ID NO: 12) B*0801(53) FIV VPRRHIRRV44 67 B*0702(20); B7(1); B8(1) B7; B8 (SEQ ID NO: 19) B*0801(124) A*1101(A3) HIV IIATDIQTK A1101(404); A3(3) A3 (SEQ ID NO: 10) A*6801(204) SIVILATDIQTT 78 89 A*0250(10) A2(5) None (SEQ ID NO: 21) FIV QESLRIQDY 2233 B*4402(88) B44(3) None (SEQ ID NO: 22) FIV

44 78 A*1101(338); A3(2) A3 (SEQ ID NO: 23) A*6801(206) ^(a) The HIVepitope sequences are from the LANL list of the best defined CTLepitopes for HIV integrase. The SIV counterpart sequences are derivedfrom LANL SIVmm239 and the FIV counterpart sequences are derived fromGenBank (ABD16378) after aa alignment with HXB2 sequence. ^(b) Theidentity (iden.) and homology (hom.) values were obtained using EMBOSSStretcher - Pairwise Sequence Alignment(www.ebi.ac.uk/Tools/psa/emboss_stretcher/). ^(c) MHC binding for HIV,SIV and FIV counterpart epitopes were predicted using the Immune EpitopeDatabase (IEDB) MHC class I binding prediction tool(http://tools.immuneepitope.org/analyze/html/mhc_binding.html). Thematching binding alleles are shown along with their binding affinityvalues (nM) which are derived from the Artificial Neural Network (ANN)analysis, where lower values represent higher binding affinity andpotential for CD8⁺ T-cell activity. The total numbers of binding alleleswith affinity below 500 nM are shown in parenthesis next to thesupertypes. ^(d) HIV epitope with non-matching SIV and FIV (directcounterparts) is in italics and the bolded FIV epitope is an indirectcounterpart with matching alleles to the HIV epitope.

The predicted results of SIV sequences can be explained by the high aaidentity between HIV and SIV as SIV is more closely related to HIV thanFIV. However, despite the relatively lower aa identity between HIV andFIV, FIV counterpart epitopes still appear to be potentially effectiveHIV antigens (see Table 6), most likely due to the slightly higher aahomology observed between the two viruses. This finding indicates thatboth SIV and FIV epitopes could induce CTL responses in human PBMCs.Therefore, conserved SIV and FIV integrase peptides can be used asimmunogens in vitro to compare and identify conserved immune responsesgenerated by the PBMCs of HIV⁺ individuals.

TABLE 7 Phase I and IIa clinical trials of HIV CTL multi-epitopevaccine. HLA Site super- (# subjects Dose/route HIV Antigens HIV type ofEpitope (%) enrolled in Vaccine type DNA (mg), MVA (# of CTL subtypesCTL selection IFNγ^(g) Trial^(a) the study)^(b) (Regimen)^(c)(p.f.u.)^(d) epitopes)^(e) involved^(f) epitopes method respondersREF^(h) IAVI-001 UK (18) DNA (d: 0, 21) 0.1 or .05 mg/i.m. p24/p17 geneA* A2, A3, Most 78% [129, 130] IAVI-002 Kenya (18) DNA (d: 0, 21) 0, 0.1or 0.5 mg/i.m. [contains TH [A, B, C, A24, B7, common 15% [131] IAVI-003UK (8) MVA (d: 0, 21) 5 × 10⁷ p.f.u./i.d. epitopes] + D, E, F, G, B8,B27, HIV 78% [129] IAVI-004 Kenya (18) MVA (mo: 0, 1) 0 or 5 × 10⁷p.f.u./ 24 CTL epitopes H]^(j) B44 subtype in 25% [131] (mo: 0) i.d.[p24(6), pol(6), Kenya IAVI-011 Switzerland/ MVA (mo: 0, 2) 0, 5 × 10⁶,5 × 10⁷ or nef(8), Env (4)] Conserved  6% [132] UK/SA (81) 2.5 × 10⁸p.f.u./i.d., epitopes i.m. or s.c. IAVI-005 UK (9) p-DNA (d: 0, 21)^(i)0.1 or 0.5 mg/i.m. 89% [129] b-MVA 5 × 10⁷ p.f.u./i.d. IAVI-006 UK (119)p-DNA (mo: 0) 0, 0.5 or 2 mg/i.m. 12% [132] b-MVA (mo: 2, 3 0 or 5 × 10⁷p.f.u./ or 5, 6) i.d. IAVI-008 Kenya (10) p-DNA (d: 0, 21) 0.5 or 1mg/i.m. 10% [131] b-MVA (mo: 9, 10) 5 × 10⁷ p.f.u./i.d. IAVI-009 Uganda(50) p-DNA (mo: 0, 1 or 0 or 0.5 mg 1× or 15% [131] 0) 2×/i.m. b-MVA(mo: 5, 8) 0 or 5 × 10⁷ p.f.u./ i.d. IAVI-010 Kenya/UK p-DNA (mo: 0, 1)0.5 mg/i.m.  3% [132] (114) b-MVA (mo: 5, 8) 0, 5 × 10⁶, 5 × 10⁷ or 2.5× 10⁸ p.f.u./i.d. IAVI-016 UK (24) p-DNA (mo: 0, 1) 0 or 4 mg/i.m. 50%[133] b-MVA (mo: 2 or 0 or 2.5 × 10⁸ p.f.u./ 0, 1) i.d. HVTN-048 USA.DNA (mo:  0.5 mg 4×/i.m. 21 CTL epitopes A, B, C, A2, A3, Conserved  0%[134] Bostwana 0, 1, 3, 6)    2 mg 4×/i.m. [Gag(4), Pol(8), D, AE, B7Epitopes  0% (36)    4 mg 4×/i.m. Vpr(1), Nef(2), AG HLA 13% Rev(1),Env(5)] + coverage TH epitope (1 pan- DR) HVTN-056 USA (40) MEP 1 mgMEP + 50 μg 4 peptides (55 B* A1, A2, Epitope 13% [135] [peptides +adjuvant] adjuvant 3×/i.m. CTL epitopes): A3, A24, clustering (mo: 0, 1,3) Env-TH/Gag- B7, B8, on LANL CTL(5) B27, USA (40) MEP [peptides + 1 mgMEP/50 μg Gag- B58, B62  3% adjuvant + GM- adjuvant + TH/GagCTL(19) CSF](mo: 0, 1, 3) 50 ug GM-CSF Env-TH/Nef-CTL 3×/i.m. (15) Env-TH/Gag-CTL(16) ANRS France (99) Lipopeptides   50 μg 4×/i.m. 5 lipopeptide (77A1, A2, Conserved 71%^(k) [136] VAC18 (mo: 0, 1, 3, 6)  150 μg 4×/i.m.CTL epitopes, A3, A24, regions 60%^(k)  500 μg 4×/i.m. containing 7 THB7, B8, 70%^(k) epitopes): B27, [Gag1(9), B58, B62 Gag2(21), Nef1(16),Nef2(21), Pol (10)] ^(a)All trials are phase I clinical trials exceptfor the bolded trial numbers which are phase IIa (with subjects not atrisks of HIV infection); International AIDS Vaccine Initiative (IAVI);HIV Vaccine Trials Network (HVTN); Agence National de Recherche sur leSIDA (ANRS). ^(b)United Kingdom (UK); South Africa (SA); United Statesof America (USA). ^(c)Prime (p); boost (b); day (d); month (mo);modified vaccinia Ankara (MVA); multi-epitope peptide (MEP); granulocytemacrophage colony stimulating factor (GM-CSF). ^(i)Nine of the 18volunteers from IAVI-001 who were primed with HIVA-DNA agreed to receivea boost 9-14 months later. ^(d)Intramuscular immunization (i.m.);intradermal immunization (i.d.); subcutaneous immunization (s.c.).^(e)MHC class I molecules can accommodate CTL epitopes of 8 to 11 aa inlength [137]. The p24/p17 represents 73% of the Gag and contains bothCTL and T-helper epitopes. The pan-DR T-helper epitope is a 13-mer thatbinds to all common HLA-DR alleles. Each of the four peptides in the MEPvaccine is made up of both TH and CTL epitopes; T helper (TH). ^(f)TheHIV subtypes used in the vaccine. *Consensus sequence. ^(j)The CTLepitopes are present in 50-90% of HIV isolates from the differentsubtypes. ^(g)Percentage of vaccinees with detected IFNγ ELISpotresponses to the CTL epitopes. The responses were detected at differenttime points, before or after the end of the immunization schedule forthe IAVI studies; after the last immunization for HVTN 064; and afterthe 2^(nd) or 3rd vaccination (single time point) for HVTN 056.^(k)Cultured ELISpot assay results. ^(h)Reference (REF).

Example 3 Monoclonal Antibodies to HIV-1 and FIV Recombinant p24Antibodies

Monoclonal antibodies (MAbs) to HIV-1 p24 and FIV p24 were produced byimmunizing mice with recombinant HIV-1 p24 and recombinant FIV p24,respectively (Table 8). Two of seven MAbs to HIV-1 p24 (HL2309 andHL2310) were only reactive to HIV-1 p24, while remaining five MAbs werereactive to both HIV-1 and FIV p24 proteins. Two of six MAbs to FIV p24(HL2350 and HL2351) were only reactive to FIV p24, while remaining fourMAbs were reactive to both HIV-1 and FIV p24 proteins. Based on Westernblot (WB) and ELISA results (FIG. 8), the epitopes recognized by HIV-1p24-specific MAbs HL2309 and HL2310 are specific for HIV-1 and are notlikely to be specific for FIV. Hence, such HIV-1-specific epitopes canbe used in Western blot or ELISA to detect HIV-1 p24 specific antibodiesin chimera HIV-1 p24/FIV backbone vaccine (hereon called chimera HIV/FIVvaccine) immunized cats but such Western blot or ELISA should not reactwith antibodies from FIV-infected cats since HL2309 and HL2310 epitopesare specific for HIV-1 and not for FIV. Using the same concept, theepitopes recognized by FIV p24-specific MAbs HL2350 and HL2351 arespecific for FIV and are not likely to be specific for HIV-1. Therefore,HL2350 and HL2351 epitopes can be used in WB or ELISA to detectantibodies from FIV-infected cats but should not react to antibodiesfrom cats immunized with chimera HIV/FIV vaccine. HL2309, HL2310,HL2350, and HL2351 epitope peptides can be used in WB or ELISA basedassays to differentiate chimera HIV/FIV vaccinated cats fromFIV-infected cats.

TABLE 8 ELISA Reactivity of Monoclonal Antibodies to HIV-1_(UCD-1) p24and FIV_(Pet) p24 Monoclo- clone isotype/ cross-reac- nal ID numberAntigen light chain tivity** HL 2309 2B3-1F6 HIV-1 UCD-1 p24 IgG1/kappaNO HL 2310 2B3-2A4 HIV-1 UCD-1 p24 IgG1/kappa NO HL 2311 2B4-1B6 HIV-1UCD-1 p24 IgG1/kappa NO HL 2312 2B4-1E8 HIV-1 UCD-1 p24 IgG1/kappa NO HL2335 4C3 HIV-1 UCD-1 p24 IgG2b/kappa NO HL 2336 5G2 HIV-1 UCD-1 p24IgM/kappa YES HL 2322 9D6 FIV Petaluma p24 IgG1/kappa YES HL 2323 7A3FIV Petaluma p24 IgG1/kappa NO HL 2324 2G12 FIV Petaluma p24 IgG1/kappaYES HL 2348 9A12-2A3 FIV Petaluma p24 IgG1/kappa YES HL 2349 9A12-2C2FIV Petaluma p24 IgG1/kappa YES HL 2350 8B2-1E1 FIV Petaluma p24IgG1/kappa NO HL 2351 8B2-2A1 FIV Petaluma p24 IgG1/kappa NO * All mousemonoclonal antibodies (MAbs) to HIV-1 p24 are positive by ELISA andWestern blot to HIV-1 p24. Similarly, all MAbs to FIV p24 are positiveby ELISA and WB to FIV p24. **Cross-reactivity denotes reactivity ofanti-HIV-1 p24 MAbs to FIV p24 and vice versa.

Example 4

See FIGS. 11 and 12. To investigate evolutionarily conserved CTLepitopes using FIV peptides, the individual 11-16-mer peptides in thepools with high responses such as FRT3/HRT3, FRT11/HRT11, FP9/Hp10, andFp14/Hp15 are being tested with PBMCs from the peptide-pool responders.IFNγ and CD3+CD8+ T-cell proliferation responses to FIV RT peptideFRT3-3 (“KKKSGKWRMLIDFRV” (SEQ ID NO:63), 15mer) are highly conservedamong HIV+ subjects and no normal (HIV-negative) individuals responded.Since FRT3 induced perforin response in PBMCs from HIV+ subjects, FRT3-3should have excellent probability in inducing CTL responses detected byperforin.

Human PBMC Assays

Stimulants: HIV-1 p24 (Hp1-Hp18) & FIV p24 (Fp1-Fp17) peptide pools were3-4 overlapping peptides of 11-15 aa long per pool, while HIV-1 RT(HRT1-HRT21) & FIV RT (FRT1-FRT21) peptide pools 3-5 overlappingpeptides of 11-15 aa_long per pool. These peptides had an overlap of 9aa spanning the entire length of the proteins.

IFNγ-ELISpot

1.0×10⁵-2.0×10⁵ PBMCs were stimulated with peptides (15 μg peptide/well)in ELISpot plates for 18 hours in AIMS V medium (at 10% normal humanserum). The spots were counted with an ELISpot reader and adjusted tospot forming units (SFU) per 10⁶ cells.

Positive reactivity was defined at ≧70 SFU/10⁶ cells after subtractingthe background derived from non-specific peptide control or mediacontrol, and the average of 3 HIV-1 negative controls (<30 SFU/10⁶cells).

CFSE-Proliferation

2×10⁵-5×10⁵ CFSE labeled PBMCs were incubated with 15-20 μg of peptidesin 600 μt of RPMI media with 10% FBS for 5 days at 37° C. (5% CO₂).After harvesting, they were labeled with allophycocyanin (APC), APC-H7,and Pacific Blue labeled monoclonal antibodies (MAb) to human CD3, CD4,and CD8, respectively and analyzed for T-cell proliferation by flowcytometry using BD LSRII (BD Biosciences). The proliferating CD4⁺ orCD8⁺ T cell populations were defined from the CD3⁺ cell population aseither CD3⁺CD8⁺ or CD3⁺CD4⁺ T cells (mutually exclusive) with low CFSE(CFSE^(low)) staining.

Intracellular Staining (ICS)

Briefly, 1×10⁶ PBMCs (freshly isolated) are stimulated with 20 μg ofpeptides for 6 hours in a total volume of 200 μL in a 96-well plate inpresence of Golgi transport inhibitor (37° C., 5% CO₂). The cells areprocessed as previously described (Horton et al. (2007)). Cells werestained with the LIVE/DEAD® fixable yellow dye (Invitrogen, Eugene,Oreg.). The monoclonal antibodies fluorochome-conjugated to humancytokines used are: APC-H7 to CD3 (clone SK7); BD Horizon V450 to CD4(clone RPA-T4); Qdot to CD8 (clone 3B5); PE-cy7 to IFN-γ (clone 4S.B3);APC to IL-2 (clone MQ1-17H12) PerCP to perforin (clone B-D48); AlexaF1.700 to granzyme-B (clone GB11) and PE to granzyme-A (clone CB9). Theflow cytometry data is collected using BD LSRII and analyzed with theFACS DIVA software.

Population of Study

HIV-1 positive adult males and females were evaluated for IFNγ ELISpot(n=28 for p24 & n=31 for RT), CD3⁺CD4⁺ T-cell CSFE-proliferation (n=24),and CD3⁺CD8⁺ T-cell CFSE-proliferation (n=24) responses to overlappingHIV-1 p24, HIV-1 RT, FIV p24, or FIV RT peptide pools. A total of 10normal healthy (HIV-1 negative) males and females were used asuninfected control group. All patients have signed an approved IRBconsent form.

FIGS. 14A-14B. IFNγ ELISpot Responses to HIV RT (reverse transcriptase)and HIV p24 (core protein) of the Primate PBMCs.

Overlapping HIV-1 p24 (FIG. 14A) and RT (FIG. 14B) peptide pool analysesare shown for nine SIV-infected rhesus macaques and four pre-infectionmacaques. Frozen PBMC were thawed and plated at the concentration of1.4×10⁵ viable cells per mL. Peptides were used at a concentration of 15μg/mL. Each bar represents an individual primate's response in spotforming units (SFU/10⁶ PBMC) after subtraction of 2 times the mediacontrol; except for the black and red bars. The black bar represents theaverage response of the pre-infection responders (Av. n=3) and the redbar represents the average response of all 4 pre-infection samples (Av.total n=4). Since these cells were frozen for over 5 years, positiveresponses are values of ≧50 SFU. We believe that fresh(non-cryopreserved) cells will give higher responses to HIV p24 peptidepools (FIG. 14A: Hp1-Hp18) and HIV RT peptide pools (FIG. 14B:Hr1-Hr21). Various mitogens (Mito.) (concanavalin A, Staphyloccocalenterotoxin A, phytohemaglutinin A) were used since these frozen cellsdid not always respond to mitogen.

Note that only two infected macaques responded to Hp 15 while threepre-infection macaques also responded. Similarly three infected macaquesresponded to Hr11 (i.e., HRT11) while three pre-infection macaques alsoresponded. These results suggest that the PBMC from uninfected macaquesrecognize these Hp15 and Hr11. Consequently the epitopes to thesepeptide pools are cross-reacting with epitopes already present in theuninfected macaques. In contrast three infected macaques are respondingto Hr6 and two infected macaques are responding to Hr14 (i.e., HRT14),but the uninfected macaques do not respond to these peptide epitopes,indicating that recognition of these peptide pools are due to SIVinfection. The PBMCs from HIV+ subjects respond robustly to Hr6 and Hr14but those of uninfected subjects did not. Thus, Hr6 (i.e., HRT6) andHr14 may be conserved between HIV and SIV, and is currently beingevaluated for the presence of conserved CTL epitopes.

NOTE that in the text we call individual peptides in the pool accordingto the order below. In the case of FRT2, individual peptide FRT2-3 isthe same as Peptide 8 (VERLELEGKVKRA (SEQ ID NO:51)) or the third onelisted under Pool FRT2.

FIV-FC1 RT (12-17-mer) with RNASE in bold. Pool FRT1 1) AQISEKIPIVKVRMK(SEQ ID NO: 52) 2) IPIVKVRMKDPTQGPQV (SEQ ID NO: 53) 3) KDPTQGPQVKQWPL(SEQ ID NO: 54) 4) GPQVKQWPLSNEKI (SEQ ID NO: 55) 5) KQWPLSNEKIEAL (SEQID NO: 56) Pool FRT2 6) LSNEKIEALTDIVER (SEQ ID NO: 57) 7)EALTDIVERLELEGK (SEQ ID NO: 58) 8) VERLELEGKVKRA = FRT2-3 (SEQ ID NO:51) 9) ELEGKVKRADPNNPW (SEQ ID NO: 59) 10) KRADPNNPWNTPVFA (SEQ ID NO:60) Pool FRT3 11) NPWNTPVFAIKKK (SEQ ID NO: 61) 12) TPVFAIKKKSGKWRM (SEQID NO: 62) 13) KKKSGKWRMLIDFRV (SEQ ID NO: 63) 14) WRMLIDFRVLNKL (SEQ IDNO: 64) 15) IDFRVLNKLTDKGA (SEQ ID NO: 65) Pool FRT4 16) LNKLTDKGAEVQLGL(SEQ ID NO: 66) 17) KGAEVQLGLPHPAGL (SEQ ID NO: 67) 18) LGLPHPAGLKMRKQV(SEQ ID NO: 68) 19) AGLKMRKQVTVLDI (SEQ ID NO: 69) Pool FRT5 20)RKQVTVLDIGDAYF (SEQ ID NO: 70) 21) VLDIGDAYFTIPL (SEQ ID NO: 71) 22)GDAYFTIPLDPDYA (SEQ ID NO: 72) 23) TIPLDPDYAPYTAF (SEQ ID NO: 73) PoolFRT6 24) PDYAPYTAFTLPRK (SEQ ID NO: 74) 25) YTAFTLPRKNNA (SEQ ID NO: 75)26) FTLPRKNNAGPGRRY (SEQ ID NO: 76) 27) NNAGPGRRYVWCSL (SEQ ID NO: 77)Pool FRT7 28) GRRYVWCSLPQGWVL (SEQ ID NO: 78) 29) CSLPQGWVLSPLIY (SEQ IDNO: 79) 30) GWVLSPLIYQSTL (SEQ ID NO: 80) 31) SPLIYQSTLDNIL (SEQ ID NO:81) Pool FRT8 32) YQSTLDNILQPFIR (SEQ ID NO: 82) 33) DNILQPFIRQNPEL (SEQID NO: 83) 34) PFIRQNPELDIYQYM (SEQ ID NO: 84) 35) PELDIYQYMDDIYI (SEQID NO: 85) 36) YQYMDDIYIGSDLNK (SEQ ID NO: 86) Pool FRT9 37)IYIGSDLNKKEHKQK (SEQ ID NO: 87) 38) LNKKEHKQKVEELRK (SEQ ID NO: 88) 39)KQKVEELRKLLLWW (SEQ ID NO: 89) 40) ELRKLLLWWGFETPEDK (SEQ ID NO: 90) 41)WGFETPEDKLQEEPPY (SEQ ID NO: 91) Pool FRT10 42) DKLQEEPPYKWMGY (SEQ IDNO: 92) 43) EPPYKWMGYELHPL (SEQ ID NO: 93) 44) WMGYELHPLTWSI (SEQ ID NO:94) 45) ELHPLTWSIQQKQL (SEQ ID NO: 95) 46) TWSIQQKQLEIPER (SEQ ID NO:96) Pool FRT11 47) IQQKQLEIPERPTL (SEQ ID NO: 97) 48) LEIPERPTLNELQKL(SEQ ID NO: 98) 49) PTLNELQKLVGKINW (SEQ ID NO: 99) 50) LQKLVGKINWASQTI(SEQ ID NO: 100) 51) KINWASQTIPDLSIK (SEQ ID NO: 101) Pool FRT12 52)SQTIPDLSIKELTTM (SEQ ID NO: 102) 53) LSIKELTTMMRGDQR (SEQ ID NO: 103)54) TTMMRGDQRLDSIR (SEQ ID NO: 104) 55) GDQRLDSIREWTTEA (SEQ ID NO: 105)56) SIREWTTEAKKEVQK (SEQ ID NO: 106) Pool FRT13 57) TEAKKEVQKAKEAI (SEQID NO: 107) 58) EVQKAKEAIETQAQL (SEQ ID NO: 108) 59) EAIETQAQLKYY (SEQID NO: 109) 60) ETQAQLKYYDPSREL (SEQ ID NO: 110) 61) KYYDPSRELYAKLSL(SEQ ID NO: 111) Pool FRT14 62) RELYAKLSLVGPHQI (SEQ ID NO: 112) 63)LSLVGPHQICYQVYH (SEQ ID NO: 113) 64) HQICYQVYHKNPEHV (SEQ ID NO: 114)65) VYHKNPEHVLWYGKM (SEQ ID NO: 115) 66) EHVLWYGKMNRQKKK (SEQ ID NO:116) Pool FRT15 67) GKMNRQKKKAENTCDI (SEQ ID NO: 117) 68)KKAENTCDIALRACY (SEQ ID NO: 118) 69) CDIALRACYKIR (SEQ ID NO: 119) 70)ALRACYKIREESIIR (SEQ ID NO: 120) 71) KIREESIIRIGKEPI (SEQ ID NO: 121)Pool FRT16 72) IIRIGKEPIYEIPA (SEQ ID NO: 122) 73) KEPIYEIPASREAW (SEQID NO: 123) 74) EIPASREAWESNLIR (SEQ ID NO: 124) 75) EAWESNLIRSPYLKA(SEQ ID NO: 125) 76) LIRSPYLKAPPPEV (SEQ ID NO: 126) Pool FRT17 77)YLKAPPPEVEFIHAA (SEQ ID NO: 127) 78) PEVEFIHAALNIKRA (SEQ ID NO: 128)79) HAALNIKRALSMI (SEQ ID NO: 129) 80) NIKRALSMIQDTPIL (SEQ ID NO: 130)81) SMIQDTPILGAETWY (SEQ ID NO: 131) 82) PILGAETWYIDGGRK (SEQ ID NO:132) Pool FRT18 83) TWYIDGGRKQGKAAR (SEQ ID NO: 133) 84) GRKQGKAARAAYW(SEQ ID NO: 134) 85) GKAARAAYWTDTGKW (SEQ ID NO: 135) 86) AYWTDTGKWQVMEI(SEQ ID NO: 136) 87) TGKWQVMEIEGSNQK (SEQ ID NO: 137) Pool FRT19 88)MEIEGSNQKAEVQAL (SEQ ID NO: 138) 89) NQKAEVQALLLALQA (SEQ ID NO: 139)90) VQALLLALQAGPEEM (SEQ ID NO: 140) 91) ALQAGPEEMNII (SEQ ID NO: 141)92) AGPEEMNIITDSQYI (SEQ ID NO: 142) Pool FRT20 93) NIITDSQYILNII (SEQID NO: 143) 94) DSQYILNIITQQPDL (SEQ ID NO: 144) 95) NIITQQPDLMEGLW (SEQID NO: 145) 96) TQQPDLMEGLWQEVL (SEQ ID NO: 146) 97) MEGLWQEVLEEMEKK(SEQ ID NO: 147) Pool FRT21 98) EVLEEMEKKIAIFI (SEQ ID NO: 148) 99)MEKKIAIFIDWVPGH (SEQ ID NO: 149) 100) IFIDWVPGHKGI (SEQ ID NO: 150) 101)DWVPGHKGIPGNEEV (SEQ ID NO: 151) 102) KGIPGNEEVDKLCQTM (SEQ ID NO: 152)Subtype-B HIV-1-UCD1 RT (11-16-mer) with RNAse in bold. Pool HRT1 1)PISPIETVPVKLK (SEQ ID NO: 153) 2) IETVPVKLKPGM (SEQ ID NO: 154) 3)VPVKLKPGMDGPKVK (SEQ ID NO: 155) 4) PGMDGPKVKQWPL (SEQ ID NO: 156) 5)GPKVKQWPLTEEKIK (SEQ ID NO: 157) Pool HRT2 6) WPLTEEKIKALIEI (SEQ ID NO:158) 7) EKIKALIEICTEMEK (SEQ ID NO: 159) 8) IEICTEMEKEGKISK (SEQ ID NO:160) 9) MEKEGKISKIGPENPY (SEQ ID NO: 161) 10) SKIGPENPYNTPVFA (SEQ IDNO: 162) Pool HRT3 11) NPYNTPVFAIKKK (SEQ ID NO: 163) 12)TPVFAIKKKDSTKWR (SEQ ID NO: 164) 13) KKKDSTKWRKLVDFR (SEQ ID NO: 165)14) KWRKLVDFRELNKR (SEQ ID NO: 166) 15) VDFRELNKRTQDFW (SEQ ID NO: 167)Pool HRT4 16) LNKRTQDFWEVQLGI (SEQ ID NO: 168) 17) DFWEVQLGIPHPAGL (SEQID NO: 169) 18) LGIPHPAGLKKKKSV (SEQ ID NO: 170) 19) AGLKKKKSVTVLDV (SEQID NO: 171) Pool HRT5 20) KKSVTVLDVGDAYF (SEQ ID NO: 172) 21)VLDVGDAYFSVPLDK (SEQ ID NO: 173) 22) AYFSVPLDKDFRKY (SEQ ID NO: 174) 23)PLDKDFRKYTAFTI (SEQ ID NO: 175) Pool HRT6 24) FRKYTAFTIPSI (SEQ ID NO:176) 25) FTIPSTNNETPGIRY (SEQ ID NO: 177) 26) NNETPGIRYQYNVL (SEQ ID NO:178) 27) GIRYQYNVLPQGWK (SEQ ID NO: 179) Pool HRT7 28) YNVLPQGWKGSPAIF(SEQ ID NO: 180) 29) GWKGSPAIFQSSMTK (SEQ ID NO: 181) 30)AIFQSSMTKILEPFR (SEQ ID NO: 182) 31) MTKILEPFRKQNPDI (SEQ ID NO: 183)Pool HRT8 32) PFRKQNPDIVIYQYM (SEQ ID NO: 184) 33) PDIVIYQYMDDLYV (SEQID NO: 185) 34) YQYMDDLYVGSDLEI (SEQ ID NO: 186) 35) LYVGSDLEIGQHRTK(SEQ ID NO: 187) 36) LEIGQHRTKIEELR (SEQ ID NO: 188) Pool HRT9 37)HRTKIEELRQHLLRW (SEQ ID NO: 189) 38) ELRQHLLRWGFTTPDK (SEQ ID NO: 190)39) RWGFTTPDKKHQK (SEQ ID NO: 191) 40) TTPDKKHQKEPPFLW (SEQ ID NO: 192)41) HQKEPPFLWMGYELH (SEQ ID NO: 193) Pool HRT10 42) FLWMGYELHPDKWTV (SEQID NO: 194) 43) ELHPDKWTVQPIML (SEQ ID NO: 195) 44) KWTVQPIMLPEKDSW (SEQID NO: 196) 45) IMLPEKDSWTVNDI (SEQ ID NO: 197) 46) KDSWTVNDIQKLVGK (SEQID NO: 198) Pool HRT11 47) NDIQKLVGKLNWA (SEQ ID NO: 199) 48)KLVGKLNWASQIYA (SEQ ID NO: 200) 49) LNWASQIYAGIKVR (SEQ ID NO: 201) 50)SQIYAGIKVRQLCKL (SEQ ID NO: 202) 51) IKVRQLCKLLRGAKA (SEQ ID NO: 203)Pool HRT12 52) CKLLRGAKALTEVI (SEQ ID NO: 204) 53) GAKALTEVIPLTKEA (SEQID NO: 205) 54) EVIPLTKEAELELA (SEQ ID NO: 206) 55) TKEAELELAENREIL (SEQID NO: 207) 56) ELAENREILKEPVH (SEQ ID NO: 208) Pool HRT13 57)REILKEPVHGVYY (SEQ ID NO: 209) 58) KEPVHGVYYDPSKDL (SEQ ID NO: 210) 59)VYYDPSKDLIAEIQK (SEQ ID NO: 211) 60) KDLIAEIQKQGQGQW (SEQ ID NO: 212)61) IQKQGQGQWTYQIY (SEQ ID NO: 213) Pool HRT14 62) GQGQWTYQIYQEPFK (SEQID NO: 214) 63) YQIYQEPFKNLKTGK (SEQ ID NO: 215) 64) PFKNLKTGKYARMR (SEQID NO: 216) 65) KTGKYARMRGAH (SEQ ID NO: 217) 66) KYARMRGAHTNDVK (SEQ IDNO: 218) Pool HRT15 67) RGAHTNDVKQLTEAV (SEQ ID NO: 219) 68)DVKQLTEAVQKIV (SEQ ID NO: 220) 69) LTEAVQKIVTESIVI (SEQ ID NO: 221) 70)KIVTESIVIWGKTPK (SEQ ID NO: 222) 71) IVIWGKTPKFKLPI (SEQ ID NO: 223)Pool HRT16 72) KTPKFKLPIQKETW (SEQ ID NO: 224) 73) KLPIQKETWEAWW (SEQ IDNO: 225) 74) IQKETWEAWWTEYW (SEQ ID NO: 226) 75) WEAWWTEYWQATWI (SEQ IDNO: 227) 76) TEYWQATWIPEWELV (SEQ ID NO: 228) Pool HRT17 77)TWIPEWELVNTPPLV (SEQ ID NO: 229) 78) ELVNTPPLVKLWYQL (SEQ ID NO: 230)79) PLVKLWYQLEKEPI (SEQ ID NO: 231) 80) WYQLEKEPIEGAETF (SEQ ID NO: 232)81) EPIEGAETFYVDGAA (SEQ ID NO: 233) 82) ETFYVDGAANRETKL (SEQ ID NO:234) Pool HRT18 83) GAANRETKLGKAGYV (SEQ ID NO: 235) 84) TKLGKAGYVTNRGR(SEQ ID NO: 236) 85) AGYVTNRGRQKVVPL (SEQ ID NO: 237) 86) RGRQKVVPLTDA(SEQ ID NO: 238) 87) RQKVVPLTDATNQK (SEQ ID NO: 239) Pool HRT19 88)PLTDATNQKTELEAI (SEQ ID NO: 240) 89) NQKTELEAIHLAL (SEQ ID NO: 241) 90)ELEAIHLALQDSGL (SEQ ID NO: 242) 91) HLALQDSGLEVNIV (SEQ ID NO: 243) 92)DSGLEVNIVTDSQYA (SEQ ID NO: 244) Pool HRT20 93) NIVTDSQYALGIIQA (SEQ IDNO: 245) 94) SQYALGIIQAQPDK (SEQ ID NO: 246) 95) GIIQAQPDKSESELV (SEQ IDNO: 247) 96) PDKSESELVSQII (SEQ ID NO: 248) 97) ESELVSQIIEQLIKK (SEQ IDNO: 249) Pool HRT21 98) SQIIEQLIKKEKVYL (SEQ ID NO: 250) 99)LIKKEKVYLAWVPAH (SEQ ID NO: 251) 100) VYLAWVPAHKGI (SEQ ID NO: 252) 101)AWVPAHKGIGGNEQV (SEQ ID NO: 253) 102) KGIGGNEQVDKLV (SEQ ID NO: 254)103) GNEQVDKLVSSGIRK (SEQ ID NO: 255) 104) KLVSSGIRKVL (SEQ ID NO: 256)NOTE that in the text we call individual peptides in the pool accordingto the order below each pool. In the case of Fp3, individual peptideFp3-3 is the same as Peptide 10 (VQLWFTAFSANL) (SEQ ID NO:257) or thethird one listed under Pool Fp3.

FIV p24 Overlapping Peptides (subtype-A FIV-Bangston backbone withsubtype-B FIV-FC1 (tubes 47-51)) Pool Fp1  1) PIQTVNGAPQYVAL (SEQ ID NO:258)  2) TVNGAPQYVALDPKM (SEQ ID NO: 259)  3) APQYVALDPKMVSIF (SEQ IDNO: 260)  4) VALDPKMVSIFMEKA (SEQ ID NO: 261) Pool Fp2  5)PKMVSIFMEKAREGL (SEQ ID NO: 262)  6) SIFMEKAREGLGGEEV (SEQ ID NO: 263) 7) KAREGLGGEEVQLWF (SEQ ID NO: 265) Pool Fp3  8) GLGGEEVQLWFTAF (SEQ IDNO: 266)  9) GEEVQLWFTAFSANL (SEQ ID NO: 267) 10) VQLWFTAFSANL =Fp3-3(SEQ ID NO: 257) 11) LWFTAFSANLTPTDM (SEQ ID NO: 268) Pool Fp4 12)AFSANLTPTDMATLI (SEQ ID NO: 269) 13) NLTPTDMATLIMAA (SEQ ID NO: 270) 14)PTDMATLIMAAPGCA (SEQ ID NO: 271) Pool Fp5 15) ATLIMAAPGCAADK (SEQ ID NO:272) 16) IMAAPGCAADKEIL (SEQ ID NO: 273) 17) APGCAADKEILDESL (SEQ ID NO:274) Pool Fp6 18) AADKEILDESLKQL (SEQ ID NO: 275) 19) KEILDESLKQLTAEY(SEQ ID NO: 276) 20) DESLKQLTAEYDRTH (SEQ ID NO: 277) 21)KQLTAEYDRTHPPDGPR (SEQ ID NO: 278) Pool Fp7 22) YDRTHPPDGPRPLPY (SEQ IDNO: 279) 23) HPPDGPRPLPYFTAA (SEQ ID NO: 280) 24) GPRPLPYFTAAEIM (SEQ IDNO: 281) 25) PLPYFTAAEIMGIGL (SEQ ID NO: 282) Pool Fp8 26)FTAAEIMGIGLTQEQQA (SEQ ID NO: 283) 27) MGIGLTQEQQAEARF (SEQ ID NO: 284)28) LTQEQQAEARFAPAR (SEQ ID NO: 285) Pool Fp9 29) EQQAEARFAPARM (SEQ IDNO: 286) 30) AEARFAPARMQCRAW (SEQ ID NO: 287) 31) FAPARMQCRAWYLEA (SEQID NO: 288) Pool Fp10 32) RMQCRAWYLEALGKL (SEQ ID NO: 289) 33)RAWYLEALGKLAAIK (SEQ ID NO: 290) 34) LEALGKLAAIKAK (SEQ ID NO: 291) PoolFp11 35) ALGKLAAIKAKSPRA (SEQ ID NO: 292) 36) LAAIKAKSPRAVQLR (SEQ IDNO: 293) 37) KAKSPRAVQLRQGAK (SEQ ID NO: 294) Pool Fp12 38)PRAVQLRQGAKEDY (SEQ ID NO: 295) 39) VQLRQGAKEDYSSFI (SEQ ID NO: 296) 40)RQGAKEDYSSFIDRL (SEQ ID NO: 297) Pool Fp13 41) KEDYSSFIDRLFAQI (SEQ IDNO: 298) 42) DRLFAQIDQEQNTA (SEQ ID NO: 299) 43) FAQIDQEQNTAEVKL (SEQ IDNO: 300) Pool Fp14 44) DQEQNTAEVKLYLK (SEQ ID NO: 301) 45)EQNTAEVKLYLKQSL (SEQ ID NO: 302) 46) AEVKLYLKQSLSIA (SEQ ID NO: 303) 47)KLYLKQSLSIANA (SEQ ID NO: 304) Pool Fp15 48) YLKQSLSIANANPDCK (SEQ IDNO: 305) 49) LSIANANPDCKRAM (SEQ ID NO: 306) 50) ANANPDCKRAMSHLK (SEQ IDNO: 307) Pool Fp16 51) PDCKRAMSHLKPESTL (SEQ ID NO: 308) 52)AMSHLKPESTLEEKL (SEQ ID NO: 309) 53) LKPESTLEEKLRA (SEQ ID NO: 310) PoolFp17 54) PESTLEEKLRACQEV (SEQ ID NO: 311) 55) LEEKLRACQEVGSPGY (SEQ IDNO: 312) 56) RACQEVGSPGYKMQLL (SEQ ID NO: 313) Consensus Subtype-B HIV-1p24 Overlapping Peptides Pool Hp1  1) PIVQNLQGQMVHQAI (SEQ ID NO: 314) 2) NLQGQMVHQAISPRT (SEQ ID NO: 315)  3) QMVHQAISPRTLNAW (SEQ ID NO:316)  4) QAISPRTLNAWVKVV (SEQ ID NO: 317) Pool Hp2  5) PRTLNAWVKVVEEKA(SEQ ID NO: 318)  6) NAWVKVVEEKAFSPE (SEQ ID NO: 319)  7)KVVEEKAFSPEVIPM (SEQ ID NO: 320) Pool Hp3  8) EKAFSPEVIPMFSAL (SEQ IDNO: 322)  9) SPEVIPMFSALSEGA (SEQ ID NO: 323) 10) IPMFSALSEGATPQD (SEQID NO: 324) Pool Hp4 11) SALSEGATPQDLNTM (SEQ ID NO: 325) 12)EGATPQDLNTMLNTV (SEQ ID NO: 326) 13) PQDLNTMLNTVGGHQ (SEQ ID NO: 327)Pool Hp5 14) NTMLNTVGGHQAAMQ (SEQ ID NO: 328) 15) NTVGGHQAAMQMLKE (SEQID NO: 329) 16) GHQAAMQMLKETINE (SEQ ID NO: 330) Pool Hp6 17)AMQMLKETINEEAAE (SEQ ID NO: 331) 18) LKETINEEAAEWDRL (SEQ ID NO: 332)19) INEEAAEWDRLHPVH (SEQ ID NO: 333) Pool Hp7 20) AAEWDRLHPVHAGPI (SEQID NO: 334) 21) DRLHPVHAGPIAPGQ (SEQ ID NO: 335) 22) PVHAGPIAPGQMREP(SEQ ID NO: 336) Pool Hp8 23) GPIAPGQMREPRGSD (SEQ ID NO: 338) 24)PGQMREPRGSDIAGT (SEQ ID NO: 339) 25) REPRGSDIAGTTSTL (SEQ ID NO: 340)Pool Hp9 26) GSDIAGTTSTLQEQI (SEQ ID NO: 341) 27) AGTTSTLQEQIGWMT (SEQID NO: 342) 28) STLQEQIGWMTNNPP (SEQ ID NO: 343) Pool Hp10 29)EQIGWMTNNPPIPVG (SEQ ID NO: 344) 30) WMTNNPPIPVGEIYK (SEQ ID NO: 345)31) NPPIPVGEIYKRWII (SEQ ID NO: 346) Pool Hp11 32) PVGEIYKRWIILGLN (SEQID NO: 347) 33) IYKRWIILGLNKIVR (SEQ ID NO: 348) 34) WIILGLNKIVRMYSP(SEQ ID NO: 349) Pool Hp12 35) GLNKIVRMYSPTSIL (SEQ ID NO: 350) 36)IVRMYSPTSILDIRQ (SEQ ID NO: 351) 37) YSPTSILDIRQGPKE (SEQ ID NO: 352)Pool Hp13 38) SILDIRQGPKEPFRD (SEQ ID NO: 353) 39) IRQGPKEPFRDYVDR (SEQID NO: 354) 40) PKEPFRDYVDRFYKT (SEQ ID NO: 355) Pool Hp14 41)FRDYVDRFYKTLRAE (SEQ ID NO: 356) 42) VDRFYKTLRAEQASQ (SEQ ID NO: 357)43) YKTLRAEQASQEVKN (SEQ ID NO: 358) Pool Hp15 44) RAEQASQEVKNWMTE (SEQID NO: 359) 45) ASQEVKNWMTETLLV (SEQ ID NO: 360) 46) VKNWMTETLLVQNAN(SEQ ID NO: 361) Pool Hp16 47) MTETLLVQNANPDCK (SEQ ID NO: 362) 48)LLVQNANPDCKTILK (SEQ ID NO: 363) 49) NANPDCKTILKALGP (SEQ ID NO: 364)Pool Hp17 50) DCKTILKALGPAATL (SEQ ID NO: 365) 51) ILKALGPAATLEEMM (SEQID NO: 366) 52) LGPAATLEEMMTACQ (SEQ ID NO: 367) Pool Hp18 53)ATLEEMMTACQGVGG (SEQ ID NO: 368) 54) EMMTACQGVGGPGHK (SEQ ID NO: 369)55) ACQGVGGPGHKARVL (SEQ ID NO: 370) SIVmm251 RT SRT1  1) PIAKVEPVKVTLKR(SEQ ID NO: 495)  2) EPVKVTLKPGKVGPK (SEQ ID NO: 496)  3) KPGKVGPKLKQWPL(SEQ ID NO: 497)  4) PKLKQWPLSKEKIVA (SEQ ID NO: 498) SR2  5)LSKEKIVALREICEK (SEQ ID NO: 499)  6) ALREICEKMEKDGQL (SEQ ID NO: 500) 7) KMEKDGQLEEAPPTNPY (SEQ ID NO: 501)  8) EAPPTNPYNTPTFAI (SEQ ID NO:502) SRT3  9) YNTPTFAIKKKDKNK (SEQ ID NO: 503) 10) IKKKDKNKWRMLIDF (SEQID NO: 504) 11) KWRMLIDFRELNRV (SEQ ID NO: 505) 12) DFRELNRVTQDFTEV (SEQID NO: 506) SRT4 13) VTQDFTEVQLGIPH (SEQ ID NO: 507) 14) EVQLGIPHPAGLAKR(SEQ ID NO: 508) 15) HPAGLAKRKRITVL (SEQ ID NO: 509) SRT5 16)KRKRITVLDIGDAYF (SEQ ID NO: 510) 17) DAYFSIPLDEEFR (SEQ ID NO: 511) 18)SIPLDEEFRQYTAF (SEQ ID NO: 512) SRT6 19) EFRQYTAFTLPSV (SEQ ID NO: 513)20) TAFTLPSVNNAEPGK (SEQ ID NO: 514) 21) VNNAEPGKRYIYKVL (SEQ ID NO:515) 22) KRYIYKVLPQGWK (SEQ ID NO: 516) SRT7 23) KVLPQGWKGSPAIFR (SEQ IDNO: 517) 24) WKGSPAIFQYTMRHV (SEQ ID NO: 518) 25) FQYTMRHVLEPFRKA (SEQID NO: 519) 26) RHVLEPFRKANPDV (SEQ ID NO: 520) SRT8 27) FRKANPDVTLVQYM(SEQ ID NO: 521) 28) VQYMDDILIASDRR (SEQ ID NO: 522) 29) DILIASDRTDLEHDR(SEQ ID NO: 523) 30) RTDLEHDRVVLQLK (SEQ ID NO: 524) SRT9 31)DLEHDRVVLQLKEL (SEQ ID NO: 525) 32) LKELLNSIGFSTPEEK (SEQ ID NO: 526)33) GFSTPEEKFQKDPPF (SEQ ID NO: 527) 34) KFQKDPPFQWMGYEL (SEQ ID NO:528) SRT10 35) FQWMGYELWPTKWKL (SEQ ID NO: 529) 36) LWPTKWKLQKIEL (SEQID NO: 530) 37) WKLQKIELPQRETW (SEQ ID NO: 531) 38) ELPQRETWTVNDIQK (SEQID NO: 532) 39) VNDIQKLVGVLNRR (SEQ ID NO: 533) SRT11 40) VGVLNWAAQIYRRR(SEQ ID NO: 534) 41) LNWAAQIYPGIKTKH (SEQ ID NO: 535) 42) YPGIKTKHLCRLIR(SEQ ID NO: 536) 43) KHLCRLIRGKMTL (SEQ ID NO: 537) SRT12 44)LIRGKMTLTEEVQW (SEQ ID NO: 538) 45) TLTEEVQWTEMAEA (SEQ ID NO: 539) 46)VQWTEMAEAEYEENK (SEQ ID NO: 540) 471 EAEYEENKIILSQER (SEQ ID NO: 541)SRT13 48) KIILSQEQEGCYY (SEQ ID NO: 542) 49) SQEQEGCYYQEGKPL (SEQ ID NO:543) 50) YYQEGKPLEATVIK (SEQ ID NO: 544) 51) PLEATVIKSQDNQW (SEQ ID NO:545) 52) IKSQDNQWSYKIH (SEQ ID NO: 546) SRT14 53) NQWSYKIHQEDKILK (SEQID NO: 547) 54) HQEDKILKVGKFAKI (SEQ ID NO: 548) 55) KVGKFAKIKNTHTNGV(SEQ ID NO: 549) SRT15 56) KNTHTNGVRLLAHVI (SEQ ID NO: 550) 57)RLLAHVIQKIGKEAR (SEQ ID NO: 551) 58) KIGKEAIVIWGQR (SEQ ID NO: 552) 59)WGQVPKFHLPV (SEQ ID NO: 553) SRT16 60) VPKFHLPVERDVW (SEQ ID NO: 554)61) LPVERDVWEQWWTDY (SEQ ID NO: 555) 62) WEQWWTDYWQVTWI (SEQ ID NO: 556)63) DYWQVTWIPEWDFI (SEQ ID NO: 557) SRT17 64) TWIPEWDFISTPPLVR (SEQ IDNO: 558) 65) STPPLVRLVFNRR (SEQ ID NO: 559) 66) RLVFNLVKDPIEGEETY (SEQID NO: 560) SIVmm251 p24 Pool Sp1  1) PVQQIGGNYVHLPLSPR (SEQ ID NO: 561) 2) GNYVHLPLSPRTLNA (SEQ ID NO: 562)  3) SPRTLNAWVKLIEEKK (SEQ ID NO:563) Pool Sp2  4) LNAWVKLIEEKKFGA (SEQ ID NO: 564)  5) IEEKKFGAEVVPGF(SEQ ID NO: 565) Pool Sp3  6) KKFGAEVVPGFQALSEGR (SEQ ID NO: 566)  7)FQALSEGCTPYDIR (SEQ ID NO: 567) Pool Sp4  8) EGCTPYDINQMLNCV (SEQ ID NO:568)  9) YDINQMLNCVGDHQA (SEQ ID NO: 569) Pool Sp5 10) DHQAAMQIIRDIINEEA(SEQ ID NO: 570) 11) MQIIRDIINEEAADW (SEQ ID NO: 571) 12)INEEAADWDLQHPQPA (SEQ ID NO: 572) Pool Sp6 13) DLQHPQPAPQQGQLR (SEQ IDNO: 573) Pool Sp7 14) APQQGQLREPSGSDI (SEQ ID NO: 574) 15)REPSGSDIAGTTSSV (SEQ ID NO: 575) Pool Sp8 16) IAGTTSSVDEQIQWM (SEQ IDNO: 576) 17) VDEQIQWMYRQQNPI (SEQ ID NO: 577) Pool Sp9 18)MYRQQNPIPVGNIYR (SEQ ID NO: 578) 19) NPIPVGNIYRRWI (SEQ ID NO: 579) PoolSp10 20) RRWIQLGLQKCVRMY (SEQ ID NO: 580) 21) LQKCVRMYNPTNIL (SEQ ID NO:581) Pool Sp11 22) MYNPTNILDVKQGPK (SEQ ID NO: 582) Pool Sp12 23)LDVKQGPKEPFQSYV (SEQ ID NO: 583) 24) KEPFQSYVDRFYKSL (SEQ ID NO: 584)Pool Sp13 25) VDRFYKSLRAEQTDA (SEQ ID NO: 585) 26) LRAEQTDAAVKNWM (SEQID NO: 586) Pool Sp14 27) TDAAVKNWMTQTL (SEQ ID NO: 469) Pool Sp15 28)WMTQTLLIQNANPDCK (SEQ ID NO: 587) 29) IQNANPDCKLVLK (SEQ ID NO: 588)Pool Sp16 30) KGLGVNPTLEEMLTAR (SEQ ID NO: 589) Pool Sp17 31)NPTLEEMLTACQGVGGPGQK (SEQ ID NO: 590) 32) GVGGPGQKARLM (SEQ ID NO: 591)

Peptides for Mapping MAB Epitopes

Antibody FIV p24 Peptides (FB = Bangston; FC = FC1; FCS = only“PDCK” changed to FC1 Code Peptide sequence (aa-mer) FB1)PIQTVNGAPQYVALDPKMVSIFMEKAREGL (30) (SEQ ID NO: 371) FB2)QYVALDPKMVSIFMEKAREGLGGEEVQL (28) (SEQ ID NO: 372) FC2)EVQLWFTAFSANLTPTDMATLIMAAP (26) (SEQ ID NO: 373) FB3)TLIMAAPGCAADKEILDESLKQLTAEYDR (29) (SEQ ID NO: 374) FB4)SLKQLTAEYDRTHPPDGPRPLPYFTAAEIM (30) (SEQ ID NO: 375) FB5)PLPYFTAAEIMGIGLTQEQQAEARFAPARM (30) (SEQ ID NO: 376) FB6)EQQAEARFAPARMQCRAWYLEALGKLAAIK (30) (SEQ ID NO: 377) FB7)LEALGKLAAIKAKSPRAVQLRQGAKEDY (28) (SEQ ID NO: 378) FB8)VQLRQGAKEDYSSFIDRLFAQIDQEQNTA (29) (SEQ ID NO: 379) FB9)FAQIDQEQNTAEVKLYLKQSLSIANANA (28) (SEQ ID NO: 380) FB10)KQSLSIANANAECKKAMSHLKPESTLEEKL (30) (SEQ ID NO: 381) FB11)LKPESTLEEKLRACQEVGSPGYKMQLL (28) (SEQ ID NO: 382) FCS12)FAQIDQEQNTAEVKLYLKQSLSIANANPDCK (31) (SEQ ID NO: 383) FCS13)LSIANANPDCKRAMSHLKPESTLEEKLRA (29) (SEQ ID NO: 384) Code Peptidesequence (aa-mer) [Hydrophlicity] FIV-Bang p24 (30-mer) FBO1)PIQTVNGAPQYVALDPKMVSIFMEKAREGL (30) [−0.02] (SEQ ID NO: 371) FBO2)PQYVALDPKMVSIFMEKAREGLGGEEVQ (28) [−0.30] (SEQ ID NO: 385) FBO3)IFMEKAREGLGGEEVQLWFTAFSANLTPTD (30) [−0.12] (SEQ ID NO: 386) FBO4)KAREGLGGEEVQLWFTAFSANLTPTDMA (28) [−0.20] (SEQ ID NO: 387) FCO5)NLTSTDMATLIMSAPGCAADKEILDETLKQ (30) [−0.03] FC1 (SEQ ID NO: 388) FBO6)TLIMAAPGCAADKEILDESLKQLTAEYDRT (30) [−0.18] (SEQ ID NO: 389) FBO7)AAPGCAADKEILDESLKQLTAEYDRTHPPD (30) [−0.83] (SEQ ID NO: 390) FBO8)CAADKEILDESLKQLTAEYDRTHPPDAPRP (30) [−1.08] (SEQ ID NO: 391) FBO9)SLKQLTAEYDRTHPPDAPRPLPYFTAAEIM (30) [−0.64] (SEQ ID NO: 392) FBO10)RTHPPDAPRPLPYFTAAEIMGIGLTQEQQA (30) [−0.56] (SEQ ID NO: 393) FBO11)LPYFTAAEIMGIGLTQEQQAEARFAPARMQ (30) [−0.11] (SEQ ID NO: 394) FBO12)GIGLTQEQQAEARFAPARMQCRAWYLEALG (30) [−0.33] (SEQ ID NO: 395) FBO13)EARFAPARMQCRAWYLEALGKLAAIKAKSP (30) [−0.16] (SEQ ID NO: 396) FBO14)CRAWYLEALGKLAAIKAKSPRAVQLRQGAK (30) [−0.20] (SEQ ID NO: 397) FBO15)KLAAIKAKSPRAVQLRQGAKEDYSSFIDRL (30) [−0.53] (SEQ ID NO: 398) FBO16)RAVQLRQGAKEDYSSFIDRLFAQIDQEQNT (30) [−0.94] (SEQ ID NO: 399) FBO17)EDYSSFIDRLFAQIDQEQNTAEVKLYLKQS (30) [−0.76] (SEQ ID NO: 400) FBO18)FAQIDQEQNTAEVKLYLKQSLSIANANAEC (30) [−0.37] (SEQ ID NO: 401) FBO19)AEVKLYLKQSLSIANANAECKKAMSHLKPE (30) [−0.39] (SEQ ID NO: 402) FBO20)LSIANANAECKKAMSHLKPESTLEEKLRAC (30) [−0.45] (SEQ ID NO: 403) FBO21)KKAMSHLKPESTLEEKLRACQEVGSPGYKM (30) [−0.92] (SEQ ID NO: 404) FBO22)STLEEKLRACQEVGSPGYKMQLL (23) [−0.44] (SEQ ID NO: 405) HIV-1-UCD1 p24(22-30-mer) HB1) PVVQNLQGQMVHQPISPRTLNAWVKVVEEK (30) [−0.34] (SEQ ID NO:406) HB2) QMVHQPISPRTLNAWVKVVEEKAFSPEVIP (30) [−0.13] (SEQ ID NO: 407)HB3) KVVEEKAFSPEVIPMFTALSEGATPQDLNT (30) [−0.12] (SEQ ID NO: 408) HB4)SPEVIPMFTALSEGATPQDLNTMLNTVGGH (30) [−0.00] (SEQ ID NO: 409) HB5)TALSEGATPQDLNTMLNTVGGHQAAMQMLK (30) [−0.19] (SEQ ID NO: 410) HB6)DLNTMLNTVGGHQAAMQMLKETINEEAAEW (30) [−0.43] (SEQ ID NO: 411) HB7)GHQAAMQMLKETINEEAAEWDRLHPVHAGP (30) [−0.75] (SEQ ID NO: 412) HB8)ETINEEAAEWDRLHPVHAGPIAPDQMREPR (30) [−1.12] (SEQ ID NO: 413) HB9)DRLHPVHAGPIAPDQMREPRGSDIAGITST (30) [−0.64] (SEQ ID NO: 414) HB10)IAPDQMREPRGSDIAGITSTLQEQIGWMTN (30) [−0.56] (SEQ ID NO: 415) HB11)GSDIAGITSTLQEQIGWMTNNPPIPVGEIY (30) [−0.09] (SEQ ID NO: 416) HB12)LQEQIGWMTNNPPIPVGEIYKRWIILGLNK (30) [−0.22] (SEQ ID NO: 417) HB13)MTNNPPIPVGEIYKRWIILGLNKIVRMYSP (30) [−0.02] (SEQ ID NO: 418) HB14)KRWIILGLNKIVRMYSPTSILDIRQGPKEP (30) [−0.31] (SEQ ID NO: 419) HB15)IVRMYSPTSILDIRQGPKEPFRDYVDRFYK (30) [−0.72] (SEQ ID NO: 420) HB16)ETINEEAAEWDRLHPVHAGPIAPDQMREPR (30) [−1.12] (SEQ ID NO: 413) HB17)DRLHPVHAGPIAPDQMREPRGSDIAGITST (30) [−0.64] (SEQ ID NO: 414) HB18)IAPDQMREPRGSDIAGITSTLQEQIGWMTN (30) [−0.56] (SEQ ID NO: 415) HB19)GSDIAGITSTLQEQIGWMTNNPPIPVGEIY (30) [−0.09] (SEQ ID NO: 416) HB20)LQEQIGWMTNNPPIPVGEIYKRWIILGLNK (30) [−0.22] (SEQ ID NO: 417) HB21)MTNNPPIPVGEIYKRWIILGLNKIVRMYSP (30) [−0.02] (SEQ ID NO: 418) HB22)KRWIILGLNKIVRMYSPTSILDIRQGPKEP (30) [−0.31] (SEQ ID NO: 419) HB23)IVRMYSPTSILDIRQGPKEPFRDYVDRFYK (30) [−0.72] (SEQ ID NO: 420) HB24)LDIRQGPKEPFRDYVDRFYKTLRAEQASQD (30) [−1.32] (SEQ ID NO: 421) HB25)FRDYVDRFYKTLRAEQASQDVKNWMTETLL (30) [−0.83] (SEQ ID NO: 422) HB26)TLRAEQASQDVKNWMTETLLVQNANPDCKT (30) [−0.79] (SEQ ID NO: 423) HB27)VKNWMTETLLVQNANPDCKTILKALGPAAT (30) [−0.01] (SEQ ID NO: 424) HB28)VQNANPDCKTILKALGPAATLEEMMTACQG (30) [−0.02] (SEQ ID NO: 425) HB29)TLEEMMTACQGVGGPGHKARVL (22) [−0.04] (SEQ ID NO: 426)

Materials and Methods for Examples 5-11

Study Population.

Blood from HIV-1 infected subjects was obtained from the University ofCalifornia at San Francisco (UCSF), the University of South Florida inTampa, and the University of Florida Center for HIV/AIDS Research,Education and Service (UF CARES) in Jacksonville. These subjects aredistributed into three groups according to the length of infection andthe anti-retroviral therapy (ART) status (Table 9). The HIV-infected(HIV+) subjects consist of long-term survivors (LTS) who have beeninfected for more than 10 years and remain healthy withoutantiretroviral therapy (LTS/ART−); subjects with short-term infectionwithout ART (ST/ART−) and subjects on ART for various amounts of time(ART+). T-cell counts and HIV-1 RNA levels were performed by clinicallaboratories at UCSF Medical Center and UF Shands Medical Center(Gainesville, Fla.). Bloods from HIV seronegative (HIV−) samples wereobtained from LifeSouth Community Blood Centers (Gainesville, Fla.) orrandomly selected volunteers at UF. The blood collections were performedaccording to the policy and protocol approved by the InstitutionalReview Boards at UF and UCSF and processed in 2-30 hours aftercollection.

RT Overlapping Peptides.

Overlapping peptides of subtype-B HIV-1UCD1 and subtype-B FIVFC1 RTproteins and selected peptides for epitope mapping were producedinitially by SynPep (Dublin, Calif.) and later by RS Synthesis LLC(Louisville, Ky.) with similar findings. Four to five consecutivepeptides (11-16 aa long with 8-10 aa overlap) were grouped into 21pools: H1-H21 for HIV and counterparts F1-F21 for FIV. In addition, 9merand 15-16mer peptides with modified sequences were also synthesized byRS Synthesis LLC and used for peptide epitope mapping as shown in Table10.

ELISpot Assays.

Enzyme-linked immunosorbent spot assays (ELISpot) for IFNγ (R&D Systems,Minneapolis, Minn.) were performed with AIM V medium containing 5%heat-inactivated (56° C., 30 min) human serum as previously described(Abbott et al. (2012)). The PBMC from HIV+ subjects were stimulated witheither peptide pool (4-5 consecutive peptides per pool at 5 μg perpeptide) or individual peptide (15 μg/well). The peptides were 11-16 aain length with 8-10 aa overlap. The results were analyzed with anELISpot reader (MVS Pacific LLC, Minneapolis, Minn.) and adjusted tospot forming units (SFU) per 10⁶ cells, after subtraction of the averagemedium control for each subject. The PBMC from HIV+ subjects werestimulated with T-cell mitogen, phytohemaglutinin A (PHA, 5 μg/mL), aspositive control. At a positive threshold of 70 SFU, HIV− subjects hadno substantial IFNγ responses (>50 SFU) to HIV and FIV peptide pools.

Flow Cytometry (FACS) for Carboxyfluoresein Diacetate Succinimide Ester(CFSE)-Proliferation and Intracellular Cytotoxin Staining (ICS).

The CFSE-proliferation analysis was performed on PBMC according to themanufacturer's protocol (Invitrogen, Carlsbad, Calif.) and processed aspreviously described (Lichterfeld et al. (2004)). Modificationsconsisted of using 2.0-5.0×10⁵ CFSE-labeled cells stimulated for 5 days(37° C., 5% CO²) with 30 μg/well of total peptides in a pool (15 μg/wellfor individual peptide, Table 10) or 5 μg/mL PHA in AIM V mediumcontaining 25 μg/mL of gentamycin and 10% heat-inactivated human serum.Subsequently these cells were harvested and labeled with the LIVE/DEADfixable yellow dye (Invitrogen) and then treated 5 min withanti-CD16/CD32 antibody (Biolegend, San Diego, Calif.) for blockingnon-specific binding before phenotype-specific antibodies. The followingantibodies were used for the CFSE-proliferation analysis: anti-CD4 APC,anti-CD3 APC-H7, and anti-CD8 Pacific Blue (BD Biosciences, San Jose,Calif.).

The ICS analysis (Horton et al. (2007)) involved stimulating 0.5-1.0×10⁶freshly isolated PBMC for 6 h with the same peptide stimulant andculture conditions as the proliferation analysis in the presence of 1μg/mL of Golgi transport inhibitor and monensin followed by labelingwith LIVE/DEAD fixable yellow dye and then treatment with anti-CD16/CD32antibody and T-cell phenotypic antibodies. The cells were subsequentlyfixed and permeabilized with Cytofix/Cytoperm solution (BD Biosciences)before reaction with anti-cytotoxin antibodies. The antibodies consistedof anti-CD3 APC-H7, anti-CD4 BD Horizon V450, and anti-CD8 FITC followedby anti-GrzB Alexa 700 and anti-GrzA PE (all from BD Biosciences), andanti-perforin PerCP (Abcam, Boston, Mass.).

In both analyses, 1.0-2.0×10⁴ cells were fixed in phosphate-bufferedsaline (PBS) containing 2% paraformaldehyde and analyzed on BD LSRIIusing FACSDIVA Software (BD Biosciences), with a positive threshold of3% CFSE^(low) for CFSE-proliferation and 1% T cells expressing cytotoxinfor ICS. The final value for each subject was derived after subtractionof the subject's medium control and the average value ofpeptide-stimulated cells from uninfected control subjects.

Statistics.

Paired Student t-test with two-tailed distribution (SigmaPlot version11.0, San Jose, Calif.) was used to evaluate the statistical differencesbetween the results from two time points in FIG. 19. These results wereconsidered statistically different when p<0.05.

Example 5 Screening for IFNγ-Inducing Epitopes on HIV-1 and FIV RT

As a first step towards identifying the CTL-associated reactive sites onHIV-1 and FIV RT proteins, the PBMC from HIV+ subjects and HIV− subjectswere screened by ELISpot analysis for IFNγ responses to overlapping RTpeptide pools of HIV-1 and FIV. NK cells, CD3+CD4+ T-helper cells,CD3+CD4+ CTLs, and CD3+CD8+ CTLs generally produce IFNγ responses toviral peptides (Abbas et al. (2010); Soghoian et al. (2012)). In thisstudy, many HIV-1 pools induced IFNγ responses of high magnitudes abovethe threshold level (≧70 SFU) with the PBMC from HIV+ subjects (FIG.17A) but none with the PBMC from HIV− subjects (data not shown).Therefore, the viral specificity of the IFNγ responses is associatedwith HIV-1 infection. The average responder frequency for all 21 poolswas 25% (range, 4-54%) (FIG. 17E). Of all HIV peptide pools screened,pool H11 induced the highest and the most frequent IFNγ responses.

Compared to the HIV peptide-pool responses, the magnitude and thefrequency of the IFNγ responses to the FIV peptide pools were much lowerin the PBMC from HIV+ subjects (FIGS. 17B and 17F). The averageresponder frequency for all 21 pools was 17% (range, 4-69%) (FIG. 17F).A noticeable exception was observed with pool F3 which induced thehighest and the most frequent cellular responses among the FIV pools(FIGS. 17B and 17F). PBMC from only five subjects (SF08, J08, J02, J09,TP01) responded to a counterpart pool H3 (grouped data only, FIG. 17A),and remarkably PBMC from four of these subjects responded to both F3 andH3 (grouped data only, FIGS. 17A and 17B). As expected, the PBMC of HIV−subjects had no IFNγ responses to the FIV pools (data not shown).Overall, HIV pools induced much higher and more frequent IFNγ responsesthan the FIV counterparts except for pool F3. However, only a few HIVand FIV counterparts were detected by the same individual (4-5responders detected both: H3/F3, H6/F6, H7/F7, H1 l/F11, H13/F13).

The immune responses observed were present in all three clinical groups(LTS/ART−, ST/ART−, ART+). In addition these groups were notstatistically different in terms of cell counts. However, it isnoteworthy that 4 of 5 responders to pool H3 are in the ST group whichmay indicate that this response has been lost in the LTS and ART+ group,possibly due to HIV infection.

Example 6 Screening for T-Cell Proliferation Epitopes on HIV-1 and FIVRTs

The presence of strong T-cell proliferation responses to HIV antigen(s)has been associated with lower viral load and better disease outcome inHIV+ individuals (McKinnon et al. (2012)). In the current studies,CD3+CD4+ T cells (hereon CD4+ T cells) from HIV+ subjects (FIGS. 18A and18B) had fewer proliferation responses than CD3+CD8+ T cells (CD8+ Tcells) to both HIV and FIV pools (FIGS. 17C and 17D). The CD4+ T cellsfrom only 5 of 26 HIV+ subjects responded to at least one HIV pool,whereas 9 of 26 HIV+ subjects responded to at least one FIV pool (FIGS.18A and 18B). In contrast, the CD8+ T cells from 17 of 26 HIV+ subjectsresponded to the HIV pools, and 22 of 26 subjects responded to the FIVpools (FIGS. 17C and 17D).

The most striking result was the high magnitude and the high frequencyof CD8+ T-cell proliferation to the FIV pools in comparison to HIV pools(FIGS. 17C and 17D). Furthermore, the average responder frequency to allFIV pools was 17% (range, 0-54%) (FIG. 17F), which is higher than theaverage of 10% (range, 0-24%) observed with the HIV pools (FIG. 17E).Only one of the HIV pools (H11) had a responder frequency of >20% (FIG.17E), while the responder frequencies to the six FIV pools (F3, F6, F7,F11, F15, F21) were >20% (FIG. 17E). In addition, only a few HIV and FIVcounterparts were detected by the same subject (2-4 responders detectedboth: H3/F3, H6/F6, H14/F14, H15/F15, H17/F17, H19/F19), but 43% (23 of53) of the total positive proliferation responses to the HIV-1 poolswere also positive to FIV counterparts.

The above results support the view that the CD8+ T-cell proliferativeresponses to FIV pools are more robust or possibly more intact thanthose to HIV pools. This finding is clearly opposite from the results ofthe IFNγ studies where the IFNγ responses were stronger against the HIVpools than the FIV pools (FIGS. 17A and 17B). These conflicting findingsmay be partially attributed to the difference in the cell types used(PBMC versus CD8+ T cells). Moreover, 12 of 15 responders showing CD8+T-cell proliferation to the F3 pool also had robust IFNγ responses tothe F3 pool (FIGS. 17B and 17D). These findings indicate that thesesubjects recognize peptide epitope(s) that induce both responses.

Example 7 The Persistence of IFNγ and Proliferation Responses toSelected HIV and FIV Peptide Pools

Due to the ability of HIV to quickly escape from immunological pressure(Leslie et al. (2004); Troyer et al. (2009)), the PBMC from IFNγresponders (FIGS. 17A and 17B) were retested at least one year later forIFNγ responses to peptide pools H6, H11, and F3. The majority of theindividuals tested retained positive IFNγ responses at the secondtime-point to H6 (8 of 11 responders) and to F3 (11 of 14 responders)but fewer to H11 (5 of 14 responders) (FIG. 19A). The cells from a fewsubjects were retested against H6 and F3 for a third time-point anddemonstrated the persistence of the IFNγ responses to the F3 pool (4 of5) but to a lesser extent to the H6 pool (2 of 4) after 3 years (FIG.19A). More importantly, the persistence of the IFNγ responses to the F3pool demonstrated the reproducibility of this activity even though noIFNγ responses were observed to the HIV− counterpart H3 by those whowere tested after 2 years (Table 10, top for H3-3 peptide).

The CD4+ and CD8+ T-cell proliferation responses did not correlate withthe IFNγ responses in general as only a low frequency of CD4+ Tresponses were observed. However, more CD4+ T-cell responses wereobserved in the production of cytotoxins. The lack of correlationbetween IFNγ ELISpot and CD8+ T-cell proliferation responses has beendescribed before, with p24 proteins. In this case, the majority ofresponses (64%) were IFNγ+/proliferation- and only 30% of the responseswere IFNγ+/proliferation+ (Richmond et al. (2011)). Furthermore, the useof PBMC in the IFNγ analysis may have contributed to the lack ofcorrelation between IFNγ and T-cell proliferation responses. Cells suchas NK cells in PBMC are known to be high producer of IFNγ (Caligiuri(2008)) and could have also given the IFNγ responses.

In addition, the CD8+ T-cell proliferation responses to F3 pool (7 of 9responders) persisted but not to H11 pool (2 of 5) and F6 pool (1 of 4)(FIG. 19B). Overall, these results suggest a major loss of Hi 1-specificIFNγ and proliferation responses with maintenance of reactions to the F3pool. Due to the strong persistent proliferation and IFNγ responses tothe F3 pool, subsequent studies focused on the F3 peptide pool and itsfive individual peptide/epitopes.

Example 8 Identifying the Peptide Epitope(s) on F3 Region that InducesIFNγ and CD8+ T-Cell Proliferation Responses

The finding that 69% and 58% of the HIV+ subjects responded to pool F3with IFNγ production and CD8+ T-cell proliferation respectively (FIGS.17E and 17F) suggested the potential that the F3 region containsmultiple epitopes. The F3 peptide pool has five overlapping peptides of13-15mers (F3-1, F3-2, F3-3, F3-4, F3-5). All ten F3 responders tested1-2 years later had IFNγ responses to the F3-3 peptide, and oneindividual each had low IFNγ responses to individual peptides F3-1 andF3-4 (FIG. 20A). Furthermore, 4 of 22 F3 responders had IFNγ responsesto the counterpart pool H3 in the first year (FIG. 17A), but all threeof the H3 responders tested on or after the second year lost IFNγresponses to H3 and had no reactivity to any of the individual 13-15merH3 peptides (Table 10, top; 0/3 IFNγ response to H3-3). As expected, allten HIV-control subjects had no IFNγ responses to individual peptides ofboth pools F3 and H3 (data not shown).

Remarkably, the majority of IFNγ responses observed to pool F3 werespecific for the F3-3 peptide, and the highest responder frequency ofCD8+ T-cell proliferation was observed with F3-3 (5 of 8) as well;slightly lower reactions were noted with F3-2, F3-4, and F3-5 (3 of 8each) (FIG. 20B). F3-3 is therefore the predominant FIV RT peptide thatgives both IFNγ and T-cell proliferation responses.

Example 9 Characterization of CTL-Associated Activities Induced by theF3 Pool and F3-3 Peptide

One of the most important CMI activities needed to control HIV infectionis potent cytotoxicity (Betts et al. (1999)). Both CD4+ CTLs and CD8+CTLs against HIV-1 have been detected in HIV+ subjects (McDermott et al.(2012)) and in HIV− individuals immunized with a candidate HIV-1 vaccine(de Souza et al. (2012)). Although activities to H6, H11, and F6 poolswere demonstrated, our focus was on CTL-associated activities to F3 pool(FIGS. 21A-21C) and its five individual peptides (FIG. 21D). 100% (11 of11) of the F3 responders expressed at least one cytotoxin (GrzA, GrzB,or perforin) in their CD4+ or CD8+ T cells similar to the 100% (8 of 8)of H11 responders but higher than the 75% (6 of 8) of H6 responders and50% (3 of 6) of F6 responders. Hence, CTL-associated epitope(s) presenton F3 and H11 were recognized by all the subjects tested. These findingssuggest that multiple CTL epitopes may reside on each of these regions.

This study showed that all five individual F3 peptides induced GrzA,GrzB, and/or perforin in the CD4+ and/or CD8+ T cells of at least one ormore HIV+ subjects tested (FIG. 21D). Based on this finding, differentCTL-associated epitopes appear to be present within all five of theindividual F3 peptides (data not shown).

Example 10 CMI Epitopes at H3-3 and F3-3 are Conserved AmongLentiviruses

According to LANL QuickAlign analysis, the H3 pool makes up a stretch ofaa that is highly conserved among lentiviruses as it is identical to 47%of the HIV-1 RTs and 7% of the SIV RTs(hiv.lanl.gov/content/sequence/QUICK_ALIGN/QuickAlign.html). AA sequenceanalysis of all HIV and FIV counterpart pairs determined that H3/F3 hadthe second highest aa identity of 66.7% (FIG. 22A). Furthermore, aasequence analysis of the individual 13-15mer peptides shows high aasequence identity and similarity between HIV and FIV (FIG. 22B). The HIVand FIV pair with the highest similarity was shown in order of thehighest to the lowest: H3-1/F3-1 (92%), H3-2/F3-2 (81%), H3-3/F3-3(75%), H3-4/F3-4 (71%), and H3-5/F3-5 (71%). Considerable similaritiesin sequences were observed when the H3 and F3 13-15mer peptides werecompared to SIV and CAEV counterpart sequences. Based on aa sequencesimilarity, both the H3/F3 peptide-pool regions and their counterpartindividual peptides are evolutionarily conserved (FIGS. 22A and 22B).

Due to the consistently higher CMI responses to F3-3 than to the otherfour individual F3 peptides (FIG. 20), subsequent studies focused onF3-3 and its HIV-counterpart H3-3. According to LANL QuickAlignanalysis, H3-3 has an 83% and 35% aa identity with various HIV-1 and SIVsequences, respectively. H3-3 and F3-3 peptides have 69% identity and75% similarity with two gaps (FIG. 22B; Table 10, top). Even with suchsequence similarity, IFNγ and CD8+ T-cell proliferation responsesgreatly differed between these peptides (Table 10).

F3-3 differs from the H3-3 used in the current study (row 1 versus row2, Table 10) by lacking one aa (Asp on position 4 of H3-3) and havingfour aa differences at the F3-3 positions 5, 9, 11, and 15. Thecombination of a D4 deletion and three changes at K10, V12, and E16 ofH3-3 with aa identical to F3-3 resulted in IFNγ responses approachingF3-3 (Table 10, F3-3m6). The addition of D4 to F3-3 (16mer) (F3-3m2)also resulted in IFNγ responses approaching F3-3, whereas the removal ofV16 from F3-3m2, giving 15mer F3-3ml, caused a major loss in IFNγresponses and also a modest loss in CD8+ T-cell proliferation responses.Furthermore, a single aa change at F3-3 positions 9 (M9→K9; F3-3m5), 11(I11→V11; F3-3m3), or 15 (V15→E15; CAEV & MVV peptide) caused majorlosses in both IFNγ and CD8+ T-cell proliferation responses. Note thatnone of the modifications of H3-3 and the peptides tested in Table 10,induced IFNγ or T-cell proliferation responses in the PBMC or the Tcells from HIV− control subjects (data not shown).

Peptide F3-3 has high degrees of aa identity to those of ungulatelentiviruses (93%, caprine arthritis-encephalitis virus [CAEV] andMaedi-Visna virus [MVV]) (Table 10). Thus, the F3-3 sequence is greatlyconserved among lentiviruses. In this regard, the ungulate peptidecounterpart of F3-3, induced IFNγ responses in the PBMC from 1 of 9 F3-3responders tested (Table 10, top). The above results demonstrate thatthe F3-3 sequence contains evolutionarily-conserved epitope(s) thatinduces persistent CMI responses, including strong CTL-associatedactivity, even when the responses to the counterpart H3-3 are lost.

Example 11

In these studies, the CMI responses by the HIV+ subjects to FIV and HIVRT peptides or peptide pools resulted in three major observations:First, the CD8+ T-cell proliferation responses to FIV pools were morerobust with higher frequency of responders than those induced by the HIVpools (FIGS. 17E and 17F). This observation was unexpected since higherlevels of IFNγ responses were observed with HIV pools than with FIVpools. These proliferation responses to the FIV pools, especially to F3,persisted over a longer time period than those to the HIV pools tested(FIG. 19B). Thus, the few aa differences between these viruses may besufficient for the CD8+ T cells to recognize the F3 but not the H3peptides. In fact, three aa substitutions in the H3-3 aa sequence(V10→I10; K12→M12; E16→V16) with aa identical to F3-3 led toimmunological responses more consistent with that of F3-3 (Table 10).This observation clearly supports our finding that only a few aa changescan substantially alter the responses to a peptide epitope.

The robust CD8+ T-cell responses by the HIV+ subjects to FIV peptidepools suggest that these peptide regions containevolutionarily-conserved epitopes. Importantly, 23 of 53 (43%) totalpositive CD8+ T-cell proliferation responses and 40 of 166 (23%) totalpositive IFNγ responses to HIV pools were also positive for theircounterpart FIV pools (FIG. 17). This observation suggests that theT-cell response measured by CD8+ T-cell proliferation (43%) was moresuccessful at screening for evolutionarily conserved peptide epitopesthan by the IFNγ response (23%).

The second observation was the profound and persistent IFNγ and CD8+T-cell proliferation responses to pool F3 which had more responders thanto any HIV pool (FIG. 19). The pool F3 induced IFNγ responses in thePBMC from a large number (69%) of HIV+ subjects and CD8+ T-cellproliferation responses in a substantial number (58%) of these subjects.These results suggest the presence of multiple CD8+ T-cell epitopes inthe F3 region. One to three F3 responders had IFNγ or CD8+ T-cellproliferation responses to F3-1 and F3-4, and both peptide epitopesinduced CTL-associated activities (FIG. 21). In fact, the LANL databaseshows three CTL-associated epitopes (NTPVFAIKK, NK9 (SEQ ID NO:427);NTPVFAIKKK, NK10 (SEQ ID NO:428); and KLVDFRELNK, KK10 (SEQ ID NO:429))on the counterpart H3. The NK9 and NK10 sequences are identical betweenFIV and HIV-1 and are found at the carboxy-end of both 13mer peptidesF3-1 and H3-1. F3-1 only differs from H3-1 by having tryptophan (W3)instead of tyrosine (Y3) at position 3. This finding suggests that thissingle aa difference resulted in the CD8+ T-cell proliferation responseto F3-1 but not to H3-1. In the case of KK10, this epitope resides onH3-4 and differs by three aa from its direct counterpart on F3-4(mLiDFRvLNK (MK10) (SEQ ID NO:430); different aa indicated in lowercase).

The third major observation was the robust IFNγ (100%, all ten F3responders tested) and CD8+ T-cell proliferation (62%, 5 of 8) responsesto the 15mer peptide F3-3 (FIG. 20). These unusually high frequencies ofresponders to the F3-3 epitope raised a question as to whether more thanone CMI epitope resides on F3-3. In this regard, current studies, usingmodified epitopes, identified three CMI epitopes on F3-3, which were notpreviously described in LANL: KKKSGKWRMLIDFRV (KV15) (SEQ ID NO:63),WRMLIDFRV (WV9) (SEQ ID NO:431), and KWRMLIDFR (KR9) (SEQ ID NO:432)(Table 10, bottom). The largest of these epitopes (F3-3; KV15) inducecytotoxin expression, and thus, one or more of them most likely areCTL-associated epitopes. These epitopes are closely related in sequenceand evolution to ungulate lentiviruses (Table 10, top). Therefore, thesefindings indicate that the F3-3 epitopes are also evolutionarilyconserved.

The unique example of pools F3/H3 (FIG. 22) highlights the existence ofevolutionarily conserved HIV RT epitope region that is less immunogenicthan its FIV RT counterpart sequence based on pools F3/H3 and peptideF3-3/H3-3 analyses. The selection pressure against HIV in humans mayexplain the lack of responses against the HIV sequence; the samepressure may not exist in cats against FIV. The use of FIV approachshows that F3-3 region may be a great target for T-cell responses in anHIV vaccine, since both IFNγ and proliferation responses to peptide F3-3by HIV+ subjects indicate that they have previously encountered suchsequence or its variant. The approach of using FIV to identify conservedregions for an HIV vaccine is a tool that compliments most approachesfor developing a T-cell-based vaccine as in mosaic vaccines (Corey etal. (2010); Barouch et al. (2010); Santra et al. (2012)). Computationalanalyses identify potential conserved epitopes that are later tested forrelevant biological activity. These analyses have been used to selectconserved HIV/FIV sequences such as the one described for HIV/FIVintegrase (Sanou et al. (2012a)). The current FIV approachsimultaneously compares both HIV/FIV epitope sequences and immunologicalresponses.

This cross-recognition of the F3-3 epitope(s) by the HIV+ subjectsdemonstrates the polyfunctionality of the T-cell subsets tested. Threepatterns with either PBMC or T cells were observed: 1) IFNγ productionby PBMC (IFNγ/PBMC), CD8+ T-cell proliferation, and CD4+ or CD8+(CD4+/CD8+) T-cell cytotoxin expression; 2) IFNγ/PBMC and CD4+/CD8+T-cell cytotoxin expression; and 3) CD8+ T-cell proliferation andCD4+/CD8+ T-cell cytotoxin expression. These observations are importantsince polyfunctional T-cell epitopes are likely to be associated with aneffective HIV vaccine (McDermott et al. (2012); Betts et al. (2006)).Although current studies have had minimal focus on CD4+ T-cellresponses, 2 of 3 F3 responders showing CD4+ T-cell proliferation alsohad expressed CD8+ T-cell responses to F3-3 (FIG. 17D and FIG. 18B).Moreover, the CD4+ T cells from substantial numbers of F3 responders hadCTL-associated cytotoxin activities in response to pool F3 (FIG. 21). Avaccine is generally administered to HIV-naïve subjects with normal CD4+T-cell immunity. Therefore, the importance of the CD4+ T-cell responsesto F3-3 should be considered when identifying CTL-associated epitopesfor an HIV vaccine. The vaccine epitopes that induce both anti-HIV CD8+and CD4+ T responses are likely to be needed for effective vaccineprotection. These studies using FIV RT peptide pools suggest thatevolutionarily-conserved immunologic epitopes could be important for aneffective HIV vaccine.

TABLE 9 Population Characteristics Group Subject^(a) Age Gender RaceHIV^(+b) CD4/μL CD8/μL Viras Load^(c) LTS/ART J01 25 F Black   11  699 935 Undetectable J10 35 F Black   12  897  659 Undetectable J11 21 FBlack   21  564  829   932 J14 47 M Hispanic   25  722 1421   6790 SF0142 M White   16 1021  996 Undetectable SF02 44 M White   12  528  394Undetectable SF03 59 M White   24  567 1040   5500 SF08 48 M White   31 529  920   3401 SF17^(d) 56 M White   11  374 1037   2000 SF19 40 MWhite   12  292  556  40000 SF23 39 M White   10  784 1018   3160 SF2450 M White   11  675  213 Undetectable ST/ART TP01 19 F Hispanic  <1 391  583  13400 TP02 28 M White  <1 1280 1375 Undetectable J02 50 FBlack 9 mo  639 1248   710 J03 27 F Black/ 8 mo  368 1254  25700Hispanic J04 22 M Black 6 mo  537 1907   1740 J05 32 M Black 2 mo  3842202   691 J06 28 M White 6 mo  501 1110 134000 J07 26 F Black 4 mo  4481306   5120 J08 19 F Black/ 9 mo  323  932   3040 Hispanic J09 27 MWhite 2 mo  482  882 109168 J12 26 M Pacific 5 mo  352  688 405000Islander J13 41 F Black 2 mo  513  632  22100 ART⁺ SF04 55 M White   28 540  864 Undetectable SF07 65 M White   25  610 2170 Undetectable SF1650 M White    8  827 1018 Undetectable SF18 38 M White    4 1082 1298  1500 SF20 53 M White   23  76 1172 Undetectable SF22 48 M White   131205  517 Undetectable J15 56 F Black   17  291 1101 Undetectable J16 48F White   21 1250 NA^(d) Undetectable HIV⁻ NB1 NA^(e) M Unknown NB2NA^(e) F Unknown NB3 NA^(e) F Unknown N1 58 F Asian N2 27 M Black N3 27F Hispanic N4 24 M Asian N5 36 M Black N6 19 F White N7 27 F Black/Hispanic Table 9 Footnote ^(a)HIV+ subjects from UF at Jacksonville (J),UCSF (SF), and University of South Florida at Tampa (TP); normal bloodfrom blood bank (NB); normal blood from UF (N). ^(b)Number of years ofHIV infection or in months (mo). ^(c)Virus load shown as copies/mL;undetectable at either <50 or <75. ^(d)Subject started ART during thestudy. ^(e)NA: not available. See Materials and Methods for otherabbreviations.

TABLE 10 Variation of H3-3/F3-3 aa sequences and immunological responses% CD8⁺ T Average SFU of IFNγ Proliferation Virus [range] [range](Subtype)^(a) 9mer or 15mer Sequence^(b) SEQ ID NO:(positive/total)^(cd) (positive/total)^(cd) Evolutionary epitopes: HIV-1H3-3 KKKdStKWRkLvDFR 165 0 [0] (0/3) 0 [0] (0/1) FIV F3-3 KKKSGKWRMLIDFRV 63 536 [105-2500] (13/13) 5.4 [0-15] (6/9) HIV-1 (C) KKKStKWRkLvDFRe 433 7 [0-37] (0/9) 0 [0] (0/5) HIV-1 KKn StKWRkLvDFRe 434 0[0] (0/9) 0.9 [0-4] (1/5) (A, B, C, D, 01_AE) SIVcpz-_(Pts)KKKdStKWRkLvDFRe^(e) 435 0 [0] (0/4) 0 [0] (0/4) CAEV & MVV KKKSGKWRMLIDFRe^(f) 436 28 [0-100] (1/9) 0.2 [0-0.8] (0/5) Modifications ofF3-3:^(g) HIV-1 H3-3 KKKdStKWRkLvDFR 165 0 [0] (0/3) 0 [0] (0/1) FIVF3-3m1 KKKdSGKWRMLIDFR 437 11 [0-45] (0/9) 0.7 [0-3.2] (1/5) FIV F3-3m2KKKdSGKWRMLIDFRV^(e) 438 280 [0-1208] (7/9) 2.0 [0-5.6] (2/5) FIV F3-3m3KKK SGKWRMLvDFRV 439 7 [0-22] (0/9) 0 [0] (0/5) FIV F3-3m4 KKKStKWRkLIDFRV 440 9 [0-60] (0/9) 0.2 [0-0.9] (0/5) FIV F3-3m5 KKKSGKWRkLIDFRV 441 20 [0-52] (0/9) 0.3 [0-1.6] (0/5) FIV F3-3m6 KKKStKWRMLIDFRV 442 251 [20-1254] (7/9) 7.0 [0-29.1] (2/5) FIV F3-3 KKKSGKWRMLIDFRV 63 536 [105-2500] (13/13) 5.4 [0-15] (6/9) Epitopes onF3-3:^(h) FIV F3-3 (KV15) KKK SGKWRMLIDFRV 63 536 [105-2500] (13/13) 5.4[0-15] (6/9) FIV 3-3 (WV9) WRMLIDFRV 431 278 [0-1295] (8/11) 1.3[0-4.2] (1/6) FIV3-3 (KR9) KWRMLIDFR 432 20 [0-70] (1/9) 0 [0] (0/6)Table 10 footnotes: ^(a)Genbank numbers as follows: HIV-1 H3-3(K03455.1); FIV F3-3 (DQ365597.1); HIV-1 (C) (FJ595343); HIV-1 (A, B, C,D, 01_AE) (AJ313415, HM035584, HQ012309, HQ586068, HE590997); SIVcpz-Pts(ACM63211); CAEV (AAG48629.1); MVV (CAC44543); HERV-K (ABA28284).^(b)Lower case letter aa different from FIV F3-3. Many HIV-1 strainshave glutamate (E) immediately after the carboxyl end of H3-3. ^(c)Usedonly responders from FIGS. 17-19; range of IFNγ responses in SFU[range]; positive responses over total tested (positive/total).^(d)Small total participant numbers due to the use of only the F3 or H3responders who are still positive during the second or third time-point.Only cells from H3 responders were used to test peptide H3-3; whilecells from F3 responders were used to test other peptides. ^(e)Only16mer sequences. ^(f)Replacing V15 with E15 in modifications F3-3m3 toF3-3m6 resulted in almost total loss of both IFNγ and proliferationresponses. ^(g)Six modifications of 15-16mer F3-3 sequences (F3-3m1 toF3-3m6) with aa present on H3-3. ^(j)Sequence designation shown inparenthesis with the first and the last aa followed by the number of aa.

FIV RT Peptide-Pool F3 NPWNTPVFAIKKKSGKWRMLIDFRVVLNKLTDKGA (SEQ ID NO:443) NPYNTPVFAIKKKDSTKWRKLVDFRELNKRTQDFW (SEQ ID NO: 23) HIV RTPeptide-Pool H3 FIV RT peptides for Pool F3: F3-1: NPWNTPVFAIKKK (13 aa)(SEQ ID NO: 61) F3-2: TPVFAIKKKSGKWRM (15) (SEQ ID NO: 62) F3-3:KKKSGKWRMLIDFRV (15) (SEQ ID NO: 63) F3-4: WRMLIDFRVLNKL (13) (SEQ IDNO: 64) F3-5: IDFRVLNKLTDKGA (14) (SEQ ID NO: 65) HIV-1 RT peptides forPool H3: H3-1: NPYNTPVFAIKKK (13) (SEQ ID NO: 163) H3-2: TPVFAIKKKDSTKWR(15) (SEQ ID NO: 164) H3-3: KKKDSTKWRKLVDFR (15) (SEQ ID NO: 165) H3-4:KWRKLVDFRELNKR (14) (SEQ ID NO: 166) H3-5: VDFRELNKRTQDFW (14) (SEQ IDNO: 167) Combine sequence of Pool F6 immediately below:PDYAPYTAFTLPRKNNAGPGRRYVWCSL (SEQ ID NO: 444)FRKYTAFTIPSTNNETPGIRYQYNVLPQGWK (SEQ ID NO: 445) Combined sequence ofPool H6 immediately above: Peptides in pool F6 F6-1: PDYAPYTAFTLPRK (14)(SEQ ID NO: 74) F6-2: YTAFTLPRKNNA (12) (SEQ ID NO: 75) F6-3:FTLPRKNNAGPGRRY (15) (SEQ ID NO: 76) F6-4: NNAGPGRRYVWCSL (14) (SEQ IDNO: 77) Peptides in pool H6 H6-1: FRKYTAFTIPSI (12) (SEQ ID NO: 176)H6-2: FTIPSTNNETPGIRY (15) (SEQ ID NO: 177) H6-3: NNETPGIRYQYNVL (14)(SEQ ID NO: 178) H6-4: GIRYQYNVLPQGWK (14) (SEQ ID NO: 179) Combinesequence of Pool F7 immediately below: GRRYVWCSLPQGWVLSPLIYQSTLDNIL (SEQID NO: 446) YNVLPQGWKGSPAIFQSSMTKILEPFRKQNPDI (SEQ ID NO: 447) Combinedsequence of Pool H7 immediately above: Peptides in pool F7 F7-1:GRRYVWCSLPQGWVL (15) (SEQ ID NO: 78) F7-2: CSLPQGWVLSPLIY (14) (SEQ IDNO: 79) F7-3: GWVLSPLIYQSTL (13) (SEQ ID NO: 80) F7-4: SPLIYQSTLDNIL(13) (SEQ ID NO: 81) Peptides in pool H7 H7-1: YNVLPQGWKGSPAIF (15) (SEQID NO: 180) H7-2: GWKGSPAIFQSSMTK (15) (SEQ ID NO: 181) H7-3:AIFQSSMTKILEPFR (15) (SEQ ID NO: 182) H7-4: MTKILEPFRKQNPDI (15) (SEQ IDNO: 183) Combine sequence of Pool F15 immediately below:GKMNRQKKKAENTCDIALRACYKIREESIIRIGKEPI (SEQ ID NO: 448)RGAHTNDVKQLTEAVQKIVTESIVIWGKTPKFKLPI (SEQ ID NO: 449) Combined sequenceof Pool H15 immediately above: Peptides for pool F15 F15-1:GKMNRQKKKAENTCDI (16) (SEQ ID NO: 117) F15-2: KKAENTCDIALRACY (15) (SEQID NO: 118) F15-3: CDIALRACYKIR (12) (SEQ ID NO: 119) F15-4:ALRACYKIREESIIR (15) (SEQ ID NO: 120) F15-5: KIREESIIRIGKEPI (15) (SEQID NO: 121) Peptides for pool H15 H15-1: RGAHTNDVKQLTEAV (15) (SEQ IDNO: 219) H15-2: DVKQLTEAVQKIV (13) (SEQ ID NO: 220) H15-3:LTEAVQKIVTESIVI (15) (SEQ ID NO: 221) H15-4: KIVTESIVIWGKTPK (15) (SEQID NO: 222) H15-5: IVIWGKTPKFKLPI (14) (SEQ ID NO: 223)

Materials and Methods for Examples 12-17

Study Population.

The blood samples of HIV⁺ subjects were obtained from the University ofCalifornia at San Francisco (UCSF) and the University of Florida Centerfor HIV/AIDS Research, Education and Service (UF CARES) in Jacksonvilleusing the protocol approved by the Institutional Review Board at UF.HIV⁺ subjects consisted of fourteen long-term survivors (LTS) who arenot receiving ART, ten subjects with short-term infection (ST) notreceiving ART, and eleven HIV⁺ subjects receiving ART. Age, gender, andrace as well as the viral and immune status of the HIV⁺ subjects used inthe current study are outlined in Table 11. The blood samples wereprocessed within 48 hours of collection. T-cell phenotyping and HIV-1load were performed by the clinical laboratories at the UCSF MedicalCenter and UF CARES. The samples from twenty-two healthy HIVseronegative (HIV−) subjects were obtained from LifeSouth CommunityBlood Centers (Gainesville, Fla.) or from UF.

ELISpot Assays.

Human enzyme-linked immunosorbent spot assays (ELISpot) (R&D Systems,Cat#XEL285) which measure IFNγ production were performed (Abbott et al.2011). The positive threshold for human IFNγ responses was >50 spotforming units (SFU)/10⁶ cells. The final value for each subject wasderived after subtracting the result of each HIV⁺ subject with the mediacontrol followed by subtraction with the average response of the HIV−subjects which was rarely more than 10 SFU.

Flow Cytometry (FACS) for Measuring CFSE-Proliferation and IntracellularCvtokine Staining (ICS).

Carboxyfluorescein diacetate succinimide ester (CFSE)-proliferationanalysis was performed according to the manufacturer's protocol(Invitrogen) and processed as previously described (Lichterfeld et al.2004) using the following modification: 2.5-5.0×10⁵ CFSE-labeled PBMCstimulated for 4-5 days (37° C., 5% CO₂) with 15-30 μg of peptides inculture media (AIM V medium, 25 g/mL gentamycin, and 10%heat-inactivated fetal bovine serum). The ICS analysis was performed aspreviously described (Horton et al. 2007; Pattacini et al. 2012).

The antibodies used for the proliferation analysis consisted of anti-CD4allophycocyanin (APC) anti-CD3 APC-H7, and anti-CD8 Pacific Blue, andthose for ICS were anti-CD3 APC-H7, anti-CD4 BD Horizon V450, anti-CD8FITC, anti-granzyme B (GrzB) Alexa 700, anti-granzyme A (GrzA) PE (BDBiosciences, Cat#555349, 560176, 558207, 560345, 555366, 560213,558904), and anti-perforin PerCP (Abcam, Cat# ab86319). Both analyseswere performed with BD LSRII and FACSDIVA™ Software (BD Biosciences),using a positive threshold of >1% CFSE^(low) for CFSE-proliferationexcept for ICS studies with threshold of >0.1% T cells expressingcytotoxin. The final value for each subject was derived aftersubtracting the result of each HIV⁺ subject with the media controlfollowed by subtraction with the average response of the media-controlsubtracted HIV-subjects.

Human Leukocyte Antigen (HLA) Analyses.

The affinity of peptide binding to HLA was determined by NetMHC version3.2 for HLA class-I (cbs.dtu.dk/services/NetMHC/), NetMHCII version 2.2for HLA class-II (cbs.dtu.dk/services/NetMHCII/), and NetCTL version 1.2for CTL-associated epitopes (cbs.dtu.dk/services/NetCTL/). The LANLdatabase for CD8⁺ and CD4⁺ epitopes are based on the HIV-1 HXB2 sequenceand identifies the epitope-interacting HLA allele(s).

Statistical Analysis.

Statistically significant differences between the results from two timepoints were calculated using a paired Student t-test with a two-taileddistribution (SigmaPlot version 11.0) and were considered statisticallysignificant when p<0.05.

Example 12 Determining Conserved Cell-Mediated Immune (CMI) PeptidesBased on IFNγ Responses

The PBMC from the 31 HIV⁺ subjects developed robust IFNγ responses tothe full length HIV-1 p24 peptide pools (FIG. 23A, 144 total responses),whereas minimal to no responses were observed with the PBMC fromHIV-negative (HIV−) control subjects (range of 0-15 spot forming unit(SFU)). The highest responder frequencies were observed to human p24peptide pool 3 (Hp3) (18 of 31; 58%) followed by Hp2, Hp10, and Hp15 (11of 31 each; 35%). A lower number of subjects responded to Hp7 and Hp14(10 of 31 each; 32%). In addition, PBMC from the HIV⁺ subjects producedIFNγ responses of low magnitudes and frequencies to all FIV p24 peptidepools (FIG. 23B, 53 total responses) except for feline p24 peptide pools(Fp)13 and Fp14 which correspond to HIV sequences within Hp14 and Hp15peptide pools. The highest responder frequencies were observed to Fp14(11 of 31; 35%) with lower responder frequency to Fp13 (5 of 31; 16%).These findings suggested that these FIV pools contain potentialcross-reactive epitopes to HIV. In addition, overlapping SIV p24 peptidepool (Sp) analysis identified a moderate number of responses to thecorresponding SIV peptide pool Sp14 (5 of 15; 33%) (FIG. 23C), thecounterpart to Fp14 and Hp15. These results suggest that evolutionarilyconserved, cross-reactive epitope(s) may reside on Hp15, Fp14, and Sp14.

Example 13 Conserved CMI Peptides Based on T-Cell ProliferationResponses

The CD8⁺ T cells of the HIV⁺ subjects proliferated more frequently andat higher magnitudes to the HIV p24 peptide pools (FIG. 24A) than to theCD4⁺ T cells (FIG. 24D) (36 CD8⁺ T-cell responses vs. 12 CD4⁺ T-cellresponses). The highest frequency of CD8⁺ T-cell proliferation responsesto HIV p24 was against pools Hp1 (6 of 27; 22%) and Hp10 (8 of 27; 30%)followed by Hp9 (4 of 27; 15%) (FIG. 24A). The same analysis performedon T cells from healthy HIV− subjects indicated that their CD4⁺ and CD8⁺T cells did not significantly recognize HIV p24 pools except for theHp15 pool with a frequency of 42%.

All results are shown after subtraction of the average result of theresponders for each peptide pool or peptide. CD8⁺ T cells from 42% (5 of12) of the HIV− subjects had a substantial response to the Hp15 peptidepool. The number of HIV⁺ responders to Hp15 is low due to a high averageresult (28% CFSE^(low)) of HIV− responders to Hp 15 that were subtractedfrom the HIV⁺ responses (FIG. 24A). Except for the Hp15 pool (thecounterpart of the Fp14 pool), the Fp9 pool, the Hp15-3 peptide in Hp15pool, and the Fp9-3 peptide in Fp9 pool with 25-51% of the value beforesubtraction, none of the other peptide pools or peptides inducedsubstantial CD8⁺ or CD4⁺ T-cell proliferation in HIV− subjects (0-20% ofthe value before subtraction).

Twelve HIV⁺ subjects had CD8⁺ T-cell proliferation responses to FIV p24pools (FIG. 24B) compared to only five subjects with CD4⁺ T-cellproliferation responses to the same peptide pools (FIG. 24E).Remarkably, substantial CD8⁺ T-cell proliferation responses weredetected in HIV⁺ subjects to Fp9 (8 of 27; 30%) and to Fp14 (4 of 27;15%) (FIG. 24B). A lower responder frequency at a lower magnitude wasdetected in HIV-subjects to Fp9 with a minimal response to Fp14. FIG.24B shows HIV⁺ response after subtracting the average of HIV−responders. When compared with the SIV p24 pools, the Fp14-counterpartSp14 pool, but not the Fp9-counterpart Sp9 pool, was recognized by CD8⁺T cells from the HIV⁺ cohorts (FIG. 24C). In addition, CD8⁺ T cells fromHIV⁺ subjects recognized multiple SIV peptide pools at a frequency of38-54% (Sp1, Sp2, Sp4, Sp10, Sp14, Sp15) (FIG. 24C). However, Sp2, Sp4,Sp10, Sp14, and Sp15 were also recognized by CD8⁺ T cells from HIV−subjects at a low frequency of 20-30%. FIG. 24C shows the HIV responseafter subtracting the average of HIV− responders. Notably, both the Sppool and its counterpart Hp1 pool were recognized by the CD8⁺ T cellsfrom HIV⁺ subjects (FIGS. 24A and 24C).

Among the FIV peptide pools, the Fp9 pool induced strong CD8⁺ T-cellproliferation responses but few IFNγ responses, while the Fp14 poolinduced both IFNγ and CD8⁺ T-cell proliferation responses (FIGS. 23B and24B). As a result, subsequent studies focused on the Fp14 and Fp9peptide pools and their HIV counterparts, Hp15 and Hp 10, respectively.

Example 14 The Persistence of IFNγ Responses to Fp14 and CD8⁺ T-CellProliferation to Fp9

PBMC from 8 of 10 (80%) HIV⁺ subjects who initially responded to theFp14 pool retained the IFNγ response for the duration of the 2-yr studyperiod while 3 of 7 tested continued to respond in the 4th yr (FIG. 25A,left graph). Although there was no statistical difference between theIFNγ responses during the 1st yr and the 2nd yr of the seven HIV⁺subjects monitored (t1 and t2, p=0.39), a statistically significantdecrease in IFNγ response was detected when the levels were comparedbetween the 2nd yr and 4th yr (t2 and t3, p=0.035) and between theinitial time point and the 4th yr (t1 and t3, p=0.014). Similarly, thePBMC in 6 of 8 (75%) initial responders to Hp15, the counterpart forFp14, remained responsive to Hp15 through the 2nd yr but showed asubstantial declining trend in the magnitude of proliferation by the 4thyr (FIG. 25A, right graph).

CD8⁺ T cells from 7 of 9 (78%) initial Fp9 responders retained T-cellproliferation responses to Fp9 during the 2-yr monitoring period (FIG.25B, left graph). Two of the seven subjects responding to the Fp9 pool(SF17, SF19) were treated with ART during or shortly after the 2nd yrtime point (Table 11) leaving four subjects on ART and four subjects noton ART by yr 4. Three of 4 subjects on ART (SF17, SF19, SF20) maintainedstable or increased levels of proliferation responses to Fp9, while onesubject (SF18) continued to remain non-responsive. This finding suggeststhat the magnitude of response to Fp9 can improve in subjects (SF19,SF20) undergoing ART. In comparison, only 3 of 5 (60%) initialresponders retained activity against Hp10, the counterpart of Fp9,through yr 2 (FIG. 25B, right graph). This response declined inmagnitude by the 4th yr. A large majority of these responders had eithera major decrease or a loss of response to the Hp10 peptide pool, whilethe response to pools Fp9, Fp14, and Hp15 persisted for at least 2 yrand some for as long as 4 yr. The Fp9 and Hp10 pools have very littlesequence similarity (Table 12), and the response to Fp9, but not Hp10,was retained over time (FIG. 25B). Therefore, subsequent studies focusedon the Fp9, Fp14, and Hp15 peptide pools. Due to the high frequency andmagnitude of the response, Fp9 was selected for epitope mapping.

Example 15 Identifying the p24 Epitope(s) that Induce CMI Responses

The HIV Hp15 pool has three well-established CD8⁺ CTL epitopes describedin LANL database that are present within the Hp15-1a, Hp15-1c, andHp15-2/3a peptides (Table 12). These CTL epitopes have high sequencesimilarity to FIV Fp14-1b, Fp14-1a, and Fp14-3/4f respectively (Table12). Furthermore, SIV Sp14-1b and Sp14-1a have sequence similarity totheir direct counterparts Hp15-1a/Fp14-1b and Hp15-2/3a/Fp14-3/4f.Hence, these peptide epitopes show moderate to high conservation betweenspecies-specific lentiviruses.

Three to four overlapping 13-15mer peptides constitute each of thepeptide pools Fp9 (Fp9-1, Fp9-2, Fp9-3), Fp14 (Fp14-1, Fp14-2, Fp14-3,Fp14-4), and Hp15 (Hp15-1, Hp15-2, Hp15-3). Fp9-3 and Hp10-3 have an aasequence similarity of 29% and identity of 12% with four single aadifferences due to gaps (Table 12). This low degree of sequencesimilarity and identity further supports the concept that epitope(s) onFp9 are most likely not in the same location as those on Hp10. Theanalysis of individual 13-15mer peptides in the Fp9 pool indicates thatthe CD8⁺ T cells of the Fp9 responders proliferate predominantly inresponse to Fp9-3 (6 of 7) and to a lesser extent to Fp9-2 (3 of 7)(FIG. 26C). Based on this result, the 15mer Fp9-3 peptide was furtherevaluated to map specific proliferative epitope(s) using shorter (9mer)overlapping peptides.

When compared to Fp9 and Fp10 pools, Fp14 and Hp15 pools have a higheraa sequence similarity (65%) and identity (35%) with one aa differencedue to a gap (Table 12). Based on aa sequence alignment analysis, theapproximate counterpart for Hp15-1 and Hp15-2 peptides are Fp14-1 andFp14-2 peptides respectively, whereas the Hp15-3 peptide containsregions that overlap both Fp14-3 and Fp14-4 peptides. Smaller regionshave more similarity between Fp14-1 and Hp15-1 peptides (Table 12,section D) and between Fp14-4 and Hp15-3 peptides (Table 12, section B).PBMC from Fp14 responders had substantial IFNγ responses to peptideFp14-3 (6 of 9 responders) followed by peptides Fp14-1 and Fp14-4 (both3 of 9) (FIG. 26A). The majority of Hp15 responders had substantial IFNγresponses to Hp15-1 (7 of 9) and fewer responses to Hp15-3 (5 of 9)(FIG. 26B). Thus, Fp14-3 and Hp15-1 contain epitopes that inducesignificant IFNγ responses. These peptides are not counterpart FIV andHIV peptides based on aa sequence analysis and therefore are notexpressing common epitope(s). However, PBMC from three LTS responded toboth Fp14-3 and its counterpart Hp15-3, indicating a conserved CMIepitope within these peptides (FIGS. 26A and 26B; bars with *).

When specific epitope analyses of Fp9 and Fp14 regions were performed,two 9mer peptides (Fp9-3c and Fp9-3d) of the Fp9 region, differing by asingle aa in carboxyl-end or amino-end, provided the highest frequencyof a CD8⁺ T-cell proliferation response (5/6 of 9) (Table 13) but at alow magnitude (<13% CFSE^(low)) (FIG. 27). Proliferation responses toFp9-3c and Fp9-3d peptides were much lower than the levels ofproliferation observed with the Fp9 pool (FIG. 27). Furthermore, thisresult is in stark contrast to the high frequency of responders (3 of 6,50%) and the higher levels (average magnitude of 14% CFSE^(low)) of CD8⁺T-cell proliferation to the 15mer Fp9-3 peptide (FIG. 27A). Thus, the15mer, but not the 9mer Fp9-3 peptides, appears to contain the epitopethat induces the bulk of the proliferation responses of both CD8⁺ andCD4⁺ T cells (FIGS. 27A and 27C).

Similarly specific epitope analysis of the Fp14-3 region with an overlapwith the Fp14-2 and Fp14-4 regions determined that a higher frequency ofHIV⁺ subjects respond to epitopes in Fp14-3 (Fp14-3d) and Fp14-4(Fp14-3/4f, overlapping both Fp14-3 and Fp14-4) more than in Fp14-2,based on both CD8⁺ T-cell proliferation and IFNγ responses (FIGS. 27Band 27F). Since the shorter sequence of 10mer Fp14-3/4f also resides inthe larger 13mer Fp14-3d sequence (Table 13), it is still possible thatboth contain the same epitope (i.e., Fp14-3/4f). This observation issupported by in silico analysis using the HLA algorithm where thepredicted HLA A2 supertype is common among all four responders for bothFp14-3d and Fp14-3/4f (Table 14). Thus, Fp14-3/4f contains the majorepitope residing in Fp14-3 and Fp14-4 that is identified by the HLA A2supertype.

Example 16 Characterization of CTL-Associated Activity Induced by Fp9,Fp14, and Hp15 Peptides

In intracellular staining (ICS) analysis for cytotoxins, both CD8⁺ andCD4⁺ T cells expressed granzyme B (GrzB) most consistently in responseto all three peptide pools tested (Fp9, Fp14, Hp15) (FIG. 28A). 40% (2of 5) HIV− subjects had CD8⁺ T cell GrzB expression to individualpeptide pool Fp9, whereas 80% (4 of 5) responded to Fp14, and 80% (4 of5) to Hp15 (FIG. 28A). Notably, GrzB was expressed by both CD4⁺ and CD8⁺T cells from the same individuals although the overall expressionmagnitude of GzB, but not the frequency, was lower in the CD4⁺ T cells(FIG. 28B). A few of the subjects who did not have GrzB responses diddemonstrate either GrzA or perforin expression (FIG. 28A). Whenindividual 13-15mer peptides from each pool were examined, CD8⁺ T cellsfrom all five HIV− subjects responded to Fp14-3, Fp14-4, and Hp15-1,whereas up to four HIV subjects responded to Fp9-3 with the productionof one or more cytotoxins. Hence, the majority of the Fp9, Fp14, andHp15 peptides induced expression of one or more cytotoxins.

The short (9-13mer) peptides of Fp9-3, Fp14-3, and Fp14-4 from previousIFNγ and proliferation studies as well as a few additional shortpeptides (Table 13) were further tested with T cells from short-termHIV-infected subjects not on ART (ST/ART−) for production of cytotoxinsand expression of CD107a (FIGS. 28C and 28D), which are commonlyexpressed by CTLs. These short 9-13mer epitopes were produced based onthe CTL algorithm of NetCTL 1.2 and HLA algorithm of NetMHC 3.4. Theshortest peptides that predicted the highest CD8⁺ T-cell responderfrequency were 9mer peptides Fp9-3c (RMQCRAWYL) (SEQ ID NO:451) andFp9-3d (ARMQCRAWY) (SEQ ID NO:452), 10mer peptide Fp14-3/4f (KLYLKQSLSI)(SEQ ID NO:453), and 13mer peptide Fp14-3d (AEVKLYLKQSLSI) (SEQ IDNO:454) (Table 13). Peptides Fp9-3, Fp9-3c and Fp9-3d induced verylittle cytotoxin expression in CD4⁺ T cells (FIG. 28C). Among thesepeptides, Fp9-3 and Fp9-3d induced more cytotoxins in CD8⁺ T cells froma slightly larger number of ST/ART− subjects (FIG. 28D). In comparison,Fp14-4 and Fp14-3/4f peptides followed by Fp14-1b and Fp14-3/4e induceda high frequency of cytotoxin expression in both CD4⁺ and CD8⁺ T cellsfrom a majority of ST/ART− subjects (FIGS. 28C and 28D). Remarkably, theFp 14-4 and Fp 14-3/4f peptides had a higher number of cytotoxinresponses and a higher frequency of responders for both CD4+ and CD8+ Tcells than their counterparts on HIV (Hp15-3; Hp15-2/3a) and SIV(Sp14-1; Sp14-1a).

When NetCTL and NetMHC predictions were compared to the responders' HLAclass-I supertype(s), four responders to peptide Fp9-3c had thepredicted responder HLA supertype A2 (Table 14). Three of them also hadan additional HLA supertype (A1, A3, B27, or B62) predicted to have astrong binding affinity to Fp9-3c. The same analysis performed on Fp9-3ddetermined that 3 of 4 responders possessed supertype B44 while oneresponder had HLA supertype A1. Both supertypes are predicted to have astrong binding affinity to Fp9-3d. Similarly, Fp14-3/4f showed supertypeA2 as the common HLA supertype correlating with all four responders.Moreover, three more subjects had additional HLA supertypes (A1, B27, orB58) with strong predicted binding affinity for Fp14-3/4f (Table 14).Hence, the ICS and the combined NetCTL/NetMHC analyses support thepresence of CD8 T-cell epitopes on Fp9-3, Fp14-3, and Fp14-4 peptides.

Example 17

Based on IFNγ ELISpot and CFSE-proliferation analysis, the PBMC and Tcells from HIV⁺ subjects (Table 11) identified at least twocell-mediated immune (CMI) peptide epitopes in the FIV p24 pools Fp9 andFp14 that could serve as potential T-cell immunogens (FIGS. 23 and 24).These peptides were effective in the majority of subjects that had acorresponding in silico predicted HLA and did not induce substantialresponses in CD4⁺ T cells from HIV− subjects. Peptide pool Fp14 inducedrobust IFNγ production with a high frequency of responders (32%) andmoderate CD8⁺ T-cell proliferation responses (FIGS. 23B and 24B). Incontrast, peptide pool Fp9 induced robust CD8⁺ T-cell proliferation witha high frequency of responders (26%) and no IFNγ responses. Mostnotably, the immune activity induced by the Fp9 and Fp14 peptide poolswas highly reproducible and persisted for up to 4 years (FIG. 25).

Unexpectedly, SIV p24 pools induced more CD4⁺ and CD8⁺ T-cellproliferation responses than the corresponding HIV p24 pools in HIV⁺subjects (FIGS. 24A and 24C). Moreover, the attenuated CD4⁺ T-cellproliferation responses to the SIV pools were highly correlative to CD8⁺T-cell proliferation responses (FIG. 24F). In some cases, these CD4⁺ Tcells could be more sensitive to HIV-1 infection (see below). However,possibly the CD4⁺ T cells that are cross-reactive to these peptidespersist in HIV⁺ subjects and can perhaps be stimulated by theseconserved epitopes to enhance the CD8⁺ T-cell responses against HIV.

In addition to high aa sequence similarity (Table 12), the peptide poolsHp15 and Fp14 induced IFNγ responses, notably, only in PBMC from HIV⁺subjects (FIGS. 26A and 26B). Furthermore, Fp14-3 and its HIVcounterpart, Hp15-3, had a high frequency of IFNγ responders. When small9-13mer peptides from the Fp14 region were evaluated (Table 13), twooverlapping peptides within Fp14-3 and Fp14-4 (Fp14-3d, Fp14-3/4f)showed high CD8⁺ and CD4⁺ T-cell proliferation responses, low IFNγresponses in HIV⁺ subjects (FIGS. 27B, 27D, 27F) and elicited noresponse in HIV− subjects (average of <10 SFU). Thus, the epitope(s)present in peptide pools Hp15 and Fp14 are specific and likely to beevolutionarily conserved.

Peptide analysis of the Fp9 region gave a high frequency of respondersmeasured by CD8⁺ T-cell proliferation to peptides Fp9-3c and Fp9-3d buthigher CD8⁺ T-cell proliferation responses to the 15mer peptide Fp9-3(Table 13, FIGS. 27A and 27C). One concern regarding the Fp9-3 peptideis its ability to elicit non-specific CD8⁺ and CD4⁺ T-cell proliferation(mitogenic effect); mitogenic stimulation can serve as an activationsignal and could enhance HIV-1 infection (Spina et al. 1997; Stevensonet al. 1990). However, Fp9-3 peptide is not a classical T-cell mitogen(compared to PHA and concanvalin A) because it does not induce IFNγ in Tcells from either HIV⁺ or HIV− subjects (FIG. 27E). IFNγ production insome cases could enhance HIV-1 infection (Yamamoto et al. 1986; Roff etal. 2014). A percentage of CD8⁺ T cells from HIV⁻ subjects proliferatedin response to the Fp9 pool and the 15mer Fp9-3 peptide but not toFp9-3c and Fp9-3d 9mer peptides. These less mitogenic peptides have astrong algorithmic prediction for CD8⁺ T-cell activity with the mostcommon HLA supertypes A2 and B44 (Table 14) (Marsh et al. 2000).Therefore, the less mitogenic Fp9-3c and Fp9-3d peptides are likelybetter candidates as vaccine immunogens.

In this report, the CTL epitopes Hp 15-c, Hp 15-1a, and Hp15-2/3a werefurther evaluated for cytotoxin expression along with their counterpartin FIV Fp14-1a, Fp14-1b, and Fp14-3/4f, respectively (FIGS. 28C and28D). In these studies, the HIV peptides and their FIV counterparts,Hp15-1c/Fp14-1a (blue box), Hp15-1a/Fp14-1b (purple box), andHp15-2/3a/Fp14-3/4f (red box) had high cytotoxin and CD107a expression.The respective SIV counterparts Sp14-1c, Sp14-1b, and Sp14-1a had aslightly lower number of responses than either their FIV or HIV-1counterpart (FIGS. 28C and 28D).

The CTL epitope within the Hp15-1 peptide described in LANL database ispredicted to bind strongly to HLA supertype B44 (Kiepiela et al. 2007).The B44 supertype is associated with a lower incidence of HIV diseaseprogression in study subjects in South Africa, Botswana, and Zimbabwe(Leslie et al. 2010; Carlson et al. 2012). The inducers of CD8⁺ T-cellproliferation in HIV− subjects are epitopes within Hp15-3 and, to alesser extent Hp15-2, but not in the Hp15-1 peptide (FIG. 29). Thus, twoknown CTL epitopes within Hp15-1c and Hp15-1a elicited substantiallevels of cytotoxin and CD107a expression but not as high as the wellcharacterized epitope Hp15-2/3a. Hp15-2/3a induced expression in bothCD4⁺ and CD8⁺ T cells and did not stimulate T cells from HIV− subjects.As a result, it should be a better candidate for use as a vaccineimmunogen.

Among the three peptides in Fp9 pool, the 15mer Fp9-3 had the highestfrequency of responders expressing one or more cytotoxins by ICSanalysis (FIGS. 28A and 28D). Based on NetCTL analysis, this peptide canmediate CD8⁺ T-cell activity by expressing peptide-specific cytotoxin(s)and using multiple HLA supertypes (A2, B7, B8, B27, B62). This findingmakes it a strong candidate as a HIV-1 vaccine immunogen (Table 14).Similarly, the epitopes in Fp14-4 and lesser extent in Fp14-3 (FIG. 28)had the highest frequency of responders expressing cytotoxin(s) withhigh binding affinity for supertypes A2 and/or B44 (Table 14). Since theHLA B44 supertype is associated with either control of HIV infectionand/or slow progression to AIDS (Tang et al. 2011; Zhang et al. 2013;Goulder and Walker 2012) and the HLA A2 supertype is associated with lowHIV transmission (MacDonald et al. 2001a; MacDonald et al. 2001b; Liu etal. 2003), targeting these supertypes are likely beneficial in thedevelopment of an effective vaccine. In addition, a recent studycorrelated HLA A2 alleles with vaccine efficacy in the RV144 HIV vaccinetrial and highlighted the importance of HLA allotypes in developing aneffective HIV vaccine (Gartland et al. 2014). Notably, the currentobservations indicate that the cross-reactive peptides Fp9-3, Fp9-3c,Fp9-3d, Fp14-3d, and Fp14-3/4f induce CMI responses in HIV⁺ subjects andare predicted to bind with highly prevalent HLA supertypes A2 and/or B44(Table 14) (Marsh et al. 2000; Gonzalez-Galarza et al. 2011; AlleleFrequency Net Database, allelefrequencies.net, accessed Oct. 2, 2014).

Previously, we described evolutionarily conserved CD8⁺ T-cell epitopeson the FIV reverse transcriptase (Sanou et al. 2013). In the currentstudy, we have identified cross-reactive p24 epitopes that are found inboth HIV and FIV peptide sequences. These results support the existenceof an evolutionary lineage among essential proteins of inter-specieslentiviruses. Being conserved, these sequences are most likely essentialfor viral fitness, and thus less likely to mutate (Sanou et al. 2012b).

In summary, by evaluating IFNγ production, CFSE proliferation, and ICSexpression in both HIV⁺ and HIV⁻ subjects (FIG. 29), we can concludethat the large 13-15mer peptides, HIV Hp15-1 and FIV Fp14-4, and small9-10mers Hp15-2/3a, Fp14-1b, Fp14-3/4e, and Fp14-3/4f induce robust CMIresponses without mitogenic stimulation. Furthermore, since the Fp9 andFp14 epitopes possess polyfunctional activity (a combination of IFNγ,T-cell proliferation and/or cytotoxin responses), they also meritconsideration as potential immunogens for inclusion in an effectiveHIV-1 vaccine (de Souza et al. 2012; Almeida et al. 2007). Selectivelytargeting these conserved sequences and monitoring non-mitogenic, T-cellspecific responses allow the identification of conserved FIV, HIV, andSIV immunogenic peptides that could be included in an HIV vaccine forprophylaxis and immunotherapy.

TABLE 11 Description of HIV⁺ Population Group Subject ^(a) Age Gender^(b) Race HIV^(+ c) CD4/μL CD8/μL Viras Load ^(d) LTS/ART- SF01 42 MWhite 16 1021  996 Undetectable SF02 44 M White 12  528  394Undetectable SF03 59 M White 24  567 1040  5500 SF05 49 M White 22  4831242 Undetectable SF08 48 M White 31  529  920  3401 SF17 ^(ef) 56 MWhite 11  374 1037  2000 SF19 ^(ef) 40 M White 12  292  556  40000 SF2143 M White 15  800 1375  3584 SF23 ^(f) 39 M White 10  784 1018  3160SF24 ^(f) 50 M White 11  675  213 Undetectable J01 25 F Black 11  699 935 Undetectable J10 35 F Black 12  897  659 Undetectable J11 21 FBlack 21  564  829   932 J14 47 M Hispanic 25  722 1421  6790 ST/ART-J02 ^(f) 50 F Black 9 mo  639 1248   710 J03 27 F Black/ 8 mo  368 125425700 Hispanic J04 22 M Black 6 mo  537 1907  1740 J05 32 M Black 2 mo 384 2202   691 J06 ^(f) 28 M White 6 mo  501 1110 134000 J07 26 F Black4 mo  448 1306  5120 J08 19 F Black/ 9 mo  323  932  3040 Hispanic J09^(f) 27 M White 2 mo  482  882 109168 J12 ^(f) 26 M Pacific 5 mo  352 688 405000 Islander J17 51 F Black 2 mo  375 1271  2140 ART+ SF04 55 MWhite 28  540  864 Undetectable SF07 65 M White 25  610 2170Undetectable SF13 52 M White 25  304  372 Undetectable SF16 50 M White 8  827 1018 Undetectable SF18 ^(f) 38 M White  4 1082 1298  1500 SF20^(f) 53 M White 23  76 1172 Undetectable SF22 48 M White 13 1205  517Undetectable J16 48 F White 21 1250 NA ^(g) Undetectable J22 36 F Black15  391  490 Undetectable J23 50 F Black 16  949 1261 Undetectable J2441 M Black 23  202 1192 Undetectable Table 11 Footnotes: ^(a) SF prefix,HIV⁺ subject from the University of California, San Francisco. J prefix,HIV⁺ subject from the University of Florida at Jacksonville; fordefinition of subjects, see legend to FIG. 23. The results for virusload and CD4/CD8 T-cell counts are from the 1st sample obtained frompatients (yr 1). ^(b) M, male; F, female. ^(c) Duration of known HIVinfection (yr). ^(d) Virus loads are shown as RNA copies/mL;undetectable ≦75 RNA copies/mL. ^(e) HIV⁺ subject who was on ARTstarting at or shortly after yr 2. ^(f) Subjects monitored for 4 yr(FIG. 25). ^(g) NA, not available.

TABLE 12 Fp9/Fp9-3, Fp14/Fp14-3, and their counterpart aa sequences andCMI responses Peptide Pool & AA Sequence SEQ ID Compared Sequences^(b)Individual Peptide^(a) (with gap)^(b) NO. Similarity (Identity) [gap] A.Hp10/Hp10-3 vs. Fp9/Fp9-3: Hp10 Pool EQIGWMTNNPPIPVGEIYKRWII 455 Hp10 &Fp9: ** ---::    *.    : * :-- 36% (16%) [5] Fp9 PoolEQQ---AEARFAPARMQCRAWYLEA 456 Hp10-3 NPPIPVGEIYKRWII 346 Hp10-3 & Fp9-3:--  *.    : * :-- 29% (12%) [4] Fp9-3   FAPARMQCRAWYLEA 288 B.Hp15/Hp15-3 vs. Fp14/Fp14-3/Fp14-4: Hp15 Pool RAEQASQEVKNWMTETLLVQNAN457 Hp15 & Fp14:   ** : *** ::.::* : **- 65% (35%) [1] Fp14 PoolDQEQNTAEVKLYLKQSLSIANA 458 Hp15-3   VKNWMTETLLVQNAN 361 Hp15-3 & Fp14-3:--** ::.::* : --- 53% (18%) [5] Fp14-3 AEVKLYLKQSLSIA 303 Hp15-3VKNWMTETLLVQNAN 361 Hp15-3 & Fp14-4: -* ::.::* : **- 67% (27%) [2]Fp14-4  KLYLKQSLSIANA 304 C. 9mer Peptides of Fp14-3/4f vs. Hp15-2/3avs. Sp14-1c: Fp14-3/4f  KLYLKQSLS 459 Hp15-2/3a & Fp14-3/4f: -* ::.::*-70% (20%) [2] Hp15-2/3a VKNWMTETL 460 Hp15-2/3a & Sp14-1a: ******:**100% (89%) [0] Sp14-1a VKNWMTQTL 461 D. 10mer Peptides of Fp14-1a/b vs.Hp15-1c/a vs. Sp14-1c/b: Fp14-1a QEQNTAEVKL 462 Hp15-1c & Fp14-1a:  ** :*** 60% (50%) [0] Hp15-1c AEQASQEVKN 463 Hp15-1c & Sp14-1b: --*:.  ***--50% (33%) [4] Sp14-1b^(c)   QTDAAVKNWM 464 Sp14-1c    TDAAVKNWMT 465Fp14-1b QNTAEVKLYL 466 Hp15-1a & Fp14-1b: * : *** :: 70% (40%) [0]Hp15-1a QASQEVKNWM 467 Hp15-1a & Sp14-1b: *:.  ***** 80% (60%) [0]Sp14-1b^(c) QTDAAVKNWM 468 Table 12 Footnotes: ^(a)Peptide pools are nothyphenated (e.g., Fp9) while the individual large peptides have a hyphenfollowed by a number (e.g., Fp9-3) indicating the number of theoverlapping individual peptide starting from amino-end. ^(b)Alignmentsdenote identical amino acids (aa) as (*), aa with most similarity basedon charge, polarity, acid/base, and hydrophilicity/hydrophobicity as(:), those with some similarity as (.), and each gap with a (-). Theinternal gaps are due to best alignment and the external gaps are duethe length of the selected peptide or peptide pool. The percentage of aasequence similarity and identity was determined from these alignmentcriteria. ^(c)Note that Sp14 pool is a single 13mer peptide Sp14-1(TDAAVKNWMTQTL) (SEQ ID NO: 469). As a result, Sp14-1c, the counterpartSIV Sp14-1 peptide for HIV Hp15-1 peptide, is a 10mer and did notinclude the first three aa (AEQ). Whereas Sp14-1b is a 10mer withglutamine (Q) added to amino-end and threonine (T) deleted from thecarboxyl-end rather than a sequence of Sp14-1c.

TABLE 13 9-13mer T-cell epitope mapping of Fp9 and Fp14 sequences usingresponders to Fp9 or Fp14 CD8⁺ T-cell CD4⁺ T-cell Peptide Code^(a)Responder Responder (No. of aa)^(b) SEQ ID NO. Peptide Sequence^(a)Frequency (%)^(c) Frequency (%)^(c) Fp9 Peptide Pool Fp9-1 (13) 286EQQAEARFAPARM 1/7 (14) 0/7 (0) Fp9-1/2a (9) 470     EARFAPARM 5/9 (56)0/9 (0) Fp9-2 (15) 287    AEARFAPARMQCRAW 3/7 (43) 1/7 (14) Fp9-2/3b (9)471         APARMQCRA 3/9 (33) 0/9 (0) Fp9-3 (15) 288       FAPARMQCRAWYLEA 6/7 (86) 1/7 (14) Fp9-3c (9) 451           RMQCRAWYL 5/9 (56) ^(d) 4/9 (44) ^(d) Fp9-3d (9) 452          ARMQCRAWY 6/9 (67) ^(d) 3/9 (33) ^(d) Fp9-3e (10) 472          ARMQCRAWYL 3/9 (33) 1/9 (11) Fp9-3f (12) 473        APARMQCRAWYL 4/9 (44) 1/9 (11) Fp14 Peptide Pool Fp14-1 (14) 301DQEQNTAEVKLYLK 4/10 (40) 2/10 (20) Fp14-1a (10) 474  QEQNTAEVKL 0/4(0)^(e) 1/4 (25)^(e) Fp14-1b (10) 466    QNTAEVKLYL 2/4 (50)^(e) 1/4(25)^(e) Fp14-1/2a (9) 476     NTAEVKLYL 4/10 (40) 1/10 (10) Fp14-2 (15)302   EQNTAEVKLYLKQSL 3/6 / (50) 0/6 (0) Fp14-2/3b (9) 477      AEVKLYLKQ 3/10 (30) 1/10 (10) Fp14-2/3c (11) 478       AEVKLYLKQSL9/10 (90) 5/10 (50) Fp14-3 (14) 303       AEVKLYLKQSLSIA 5/6 (83) 0/6(0) Fp14-3d (13) 454       AEVKLYLKQSLSI 10/10 (100) ^(d) 7/10 (70) ^(d)Fp14-3/4e (9) 479           LYLKQSLSI 9/10 (90) 6/10 (60) Fp14-3/4f (10)453           KLYLKQSLSI 10/10 (100) ^(d) 6/10 (60) ^(d) Fp14-3/4g (9)480            YLKQSLSIA 6/10 (60) 0/10 (0) Fp14-4 (13) 304         KLYLKQSLSIANA 6/6 (100) 1/6 (17) Table 13 Footnotes: ^(a)The13-15mer peptide designations used in the peptide pools and theircorresponding aa sequences are in bold. ^(b)Number of amino acids.^(c)The responder frequencies to the large peptides were derived from yr2 and included only responders to either the Fp9 or Fp14 peptide poolvia CFSE proliferation. The responder frequencies to the small 9-13merpeptides were obtained at yr 4 and included only responders to eitherthe Fp9 or Fp14 peptide pool via both proliferation and IFNγ. ^(d)Thetwo highest frequency of responders to small peptides by both CD8⁺ andCD4⁺ T cells are highlighted in bold. ^(e)Proliferation results are fromsmall number of subjects tested (n = 4) but ICS results are from fivesubjects (FIG. 28).

TABLE 14 HLA supertypes of the responders to key short and long peptidesPeptide NetCTL NetMHC Supertypes of Algorithm and Code (No. Predic-Predic- Responder Responder Common Responder Frequency in of aa)tion^(a) tion^(a) (HLA-A/B supertypes)^(a) Supertype(s)^(b) %(magnitude) type^(c) Fp9-3 A2, B7, A2, B7, SF17 (A1/A2, B7/B7) SF17 (A2,B7) 67% (X-high) CD8 Proliferation (15) B8, B27, B8, B27 SF18 (A1/A1,B8/B44) SF18 (B8) 83% (high) CD4 Proliferation B62 SF19 (A2/A3, B44/B62)SF19 (A2, B62) 14% (medium) IFNγ SF20 (A2/A2, B44/B58) SF20 (A2) HighCytotoxins Fp9-3c A2, B8, A1, A2, SF17 (A1/A2, B7/B7) SF17 (A1, A2) 56%(low) CD8 Proliferation (9) B62 A3, B8, SF19 (A2/A3, B44/B62) SF19 (A2,B62) 44% (low) CD4 Proliferation B27 SF20 (A2/A2, B44/B58) SF20 (A2) 22%(low) IFNγ SF24 (A2, A3, B27/B44) SF24 (A2, A3, B27) Extremely LowCytotoxins Fp9-3d B27 A1, A3, SF17 (A1/A2, B7/B7) SF17 (A1) 56% (low)CD8 Proliferation (9) B27, B44 SF19 (A2/A3, B44/B62) SF19 (A3, B44) 33%(low) CD4 Proliferation SF20 (A2/A2, B44/B58) SF20 (B44) 0% (none) IFNγSF24 (A2, A3, B27/B44) SF24 (A3, B27, B44) Moderate Cytotoxins (onlyCD8) Fp14-3d A24, A1, A2, SF17 (A1/A2, B7/B7) SF17 (A1, A2) 100% (high)CD8 Proliferation (13) B44 A24, B27, SF19 (A2/A3, B44/B62) SF19 (A2,B44) 70% (low) CD4 Proliferation B44 SF20 (A2/A2, B44/B58) SF20 (A2,B44) 40% (low) IFNγ SF24 (A2, A3, B27/B44) SF24 (A2, B27, B44) LowCytotoxins (only CD8) Fp14-3/4f A24 A1, A2, SF17 (A1/A2, B7/B7) SF17(A1, A2) 100% (high) CD8 Proliferation (10) A24, B27, SF19 (A2/A3,B44/B62) SF19 (A2) 60% (low) CD4 Proliferation B58 SF20 (A2/A2, B44/B58)SF20 (A2, B58) 40% (low) IFNγ SF24 (A2, A3, B27/B44) SF24 (A2, B27) HighCytotoxins Fp14-1 A1, B39, A1, B44, SF17 (A1/A2, B7/B7) SF17 (A1) 50%(low) CD8 Proliferation (14) B58 B62 SF18 (A1/A3, B8/B44) SF18 (A1, B44)37% (low) CD4 Proliferation SF20 (A2/A2, B44/B58) SF20 (B44) 33%(medium) IFNγ SF23 (A1/A3, B27/B44) SF23 (A1, B44) High CytototoxinsHp15-1 B58 B44, B58 SF18 (A1/A1, B8/B44) SF18 (B44) 50% (low) CD8Proliferation (14) SF19 (A2/A3, B44/B62) SF19 (B44) 0% (none) CD4Proliferation SF20 (A2/A2, B44/B58) SF20 (B44, B58) 78% (medium) IFNγSF24 (A2, A3, B27/B44) SF24 (B44) High Cytotoxins Table 14 Footnotes:^(a)Four subjects who responded to the designated peptide were HLA classI typed, and their HLA alleles were compared to the HLA supertype(s)predicted for the designated peptide using the NetCTL 1.2 and NetMHC 3.2algorithms. The HLA A and HLA B allotypes for the subjects are shown asHLA supertypes. ^(b)The most common supertypes between subjects and theHLA algorithm predictions are shown. The bolded supertype represents themost common supertypes among the subjects. ^(c)All results were fromresponders to either Fp9 or Fp14 peptide pools. The results forindividual 9-11mer peptides were derived from FIG. 28. Those for13-15mer peptides (Fp9-1, Fp14-1, Hp15-1) were derived from FIGS. 5and/or 7. The average positive values are considered low when thefrequency of response is <15% CFSE or <125 SFU, and high when >30 CSFEor >300 SFU. The cytotoxin result is considered high when four or fivesubjects express one or more cytotoxins in the CD8⁺ T cells.

Example 18 Selection of Conserved FIV and HIV-1 p24 and RT Peptide Poolsand Peptides

In our recent studies, the PBMC and T cells from HIV⁺ subjects respondedto two FIV p24 peptide-pools Fp9 and Fp14 (FIG. 30) (Roff et al. 2015)and one FIV RT peptide-pool FRT3 (Sanou et al. 2013). These peptidepools were identified by IFNγELISpot of PBMC and carboxyfluoreseindiacetate succinimide ester (CFSE)-proliferation of CD3 CD4+ and CD3CD8+ T cells. FIV p24-pool Fp14 and RT-pool FRT3 induced both CD8+T-cell proliferation and IFNγ responses but minimal CD4+ T-cellproliferation. In contrast, pool Fp9 induced predominantly CD8+ T-cellproliferation but minimal CD4+ T-cell proliferation or IFNγ responses.The p24-peptide Fp14-3 of pool Fp14 induced the most IFNγ responsesfollowed by peptide Fp14-1, while the p24-peptide Fp9-3 of pool Fp9induced CD8+ T-cell proliferation. The majority of the responses toRT-pool FRT3 were specific for peptide FRT3-3. However, intracellularcytokine/cytotoxin staining (ICS) analyses determined that peptidesFp14-3, Fp14-4, and FRT3-4 induced the highest frequency and levels ofCD8+ CTL-associated activity followed by peptide Fp9-3, Fp14-1, andFRT3-1 (Fp14 peptides in FIGS. 31A-31B; FRT3 peptides in (Sanou et al.2013)). The magnitude and frequency of CTL-associated cytotoxin(perforin, granzyme A, granzyme B) responses were much higher and morefrequent in CD8+ T cells than CD4+ T cells (Roff et al. 2015; Sanou etal. 2013). This observation suggests that CD8+ CTLs are induced inresponses to FIV peptides Fp9-3, Fp14-3, and Fp14-4, and also to thecounterpart HIV-1 peptides Hp15-1 and Hp15-3. Our approach identifiedthe HIV-1 peptides Hp15-1 and Hp15-3 to be the evolutionarily orlentivirally conserved epitopes which induced CTL-associated activity inT cells.

Remarkably, prototype FIV (IWV)-vaccinated cats (Coleman et al. 2014)also responded with high magnitude and/or frequency of T-cellproliferation to pools Fp9 and Fp14 (FIGS. 32A-32B). Hence, Fp9 and Fp14pools are recognized by both HIV subjects and FIV-vaccinated cats andthus contain EC T-cell epitopes (FIGS. 30 and 32). AdditionalFIV-vaccinated semi-inbred and outbred cats were tested for Fp9 and Fp14peptide pools with similar results as in FIG. 30 (FIG. 33A). Howeverthey induced more IFNγ responses to pools Fp9 and Fp14 than thoseinduced by FIV-infected cats. Individual peptides Fp14-1, Fp14-2, andFp14-3 induced consistently low levels (e.g., 50-200 SFU considered lowlevels) of IFNγ responses in FIV-vaccinated cats (FIG. 33C, left),whereas peptide Fp14-4 induced the most CD8⁺ T-cell proliferationresponses followed by peptides Fp14-2 and Fp14-3 (FIG. 33C, right).Notably, peptides Fp14-1 and Fp14-2 induced the most CD4⁺ T-cellproliferation.

Evolutionarily Conserved (EC) or Lentivirally Conserved T-Cell Epitopes.

Above studies demonstrate that both HIV⁺ subjects and FIV-vaccinatedcats recognized certain regions of FIV (e.g., Fp9 pool) or of both FIVand HIV-1 (e.g., Fp14 and Hp15 pools; FRT3 and HRT3 pools). Moderate toidentical aa sequence similarities/identities are observed between HIV-1and FIV or SIV at conserved regions (i.e., overlapping peptide pools) ofHp15/Sp14/Fp14 (Table 15) and at conserved epitope FRT3-3 (Table 16).Notably, there is high (100%) similarity and identity (87-96%) withinHIV-1 subtypes A-D at the Hp15 region (Table 15). 100% identity isobserved among all HIV subtypes at the FRT3-3 epitope (Table 16). Thus,the Hp15/Fp14 region on p24 and HRT3-3/FRT3-3/SRT3-3 epitopes on RT arehighly conserved epitopes and considered evolutionarily conserved (EC)epitopes. The high similarity between HIV-1 and SIV strongly suggeststhe existence of more EC epitopes between these lentiviruses that inducea high CD8⁺ T-cell responder frequency in PBMC from HIV⁺ subjects (HIV-1pool Hp1 and SIV pool Sp1 FIGS. 30A, 30C) (Roff et al. 2015).Interestingly, our approach has identified additional non-conservedreactive epitopes including pool Fp9, which has minimal identity orsimilarity to the HIV and SIV counterparts and has the closestsimilarity to HIV Nef based on Los Alamos National Laboratory (LANL)QuickAlign program (53% similarity, 47% identity, 2 gaps). Theseadditional identified non-conserved sequences including this 13mer Nefcounterpart of Fp9 will also be evaluated as candidate peptide epitopefor AIDS vaccine.

Sections A and C in Table 15 show the EC epitope selection methods forHp15 peptides (Section A) and Fp14 peptides (Section C). EC peptides arefirst screened by IFNγ ELISpot analysis (IFN) and CFSE-based CD8⁺ (8P)and CD4⁺ (4P) T-cell proliferation analyses followed by CD8⁺ T-cell ICS(8C), CD4⁺ T-cell ICS (4C), and viral enhancing/inhibitory assay, andresponses in HIV negative control subjects (NC).

Multiple Antigenic Peptide (MAP) Vaccine Study with EC Epitope Peptides.

Since peptide pools and individual peptides of Fp114 and FRT3 inducedCTL-associate cytotoxin expression in T cells from HIV⁺ subjects, an invivo study was performed to test whether if vaccination with these ECepitope peptides of FIV p24 and RT can elicit protective immunityagainst FIV in laboratory cats. Eight semi-inbred cats that were primed1× with the prototype vaccine and boosted 4×-6× with 200 μg oflipophylic (Pam, palmitate C16)-MAP. The three Pam-MAPs consisted of FIVp24-peptide Fp114-1 alone (MAP1b) or together with the FIV p24-peptideFp4-3 (MAP1: Fp4-3/furin-sensitive-linker/14-1) and FIV RT peptidesFRT3-3 overlapped with FRT3-4 (MAP2) and were administered SC/ID in FD-1adjuvant with feline IL12 (FeIL12) (FIG. 34) (Table 17). Peptide Fp4-3and peptide-pool Fp4 induced predominantly IFNγ, IL2, and CD4+ T-cellproliferation responses (pool Fp4 in FIGS. 31A-31B; data not shown forFp4-3), whereas peptide Fp14-1 induced IFNγ and CD4+ T-cellproliferation responses in the PBMC from FIV-vaccinated cats (FIGS.33A-33C). Using the same assay system, FRT3-3 and FRT3-4 induced strongIL2 and CD8+ T-cell proliferation responses but modest IFNγ (FRT3-3) andnegligible CD4+ T-cell proliferation responses (data not shown). Howeverin the T cells from HIV+ subjects, Fp14-1, FRT3-3, and FRT3-4 stimulatedIFNγ production, CD8+ T-cell proliferation, and EC-CTL-associatedactivities (FIGS. 31A-31B) (Roff et al. 2015; Sanou et al. 2013), and istherefore an EC T-cell epitope. These peptides were chosen based ontheir ability to elicit FIV-specific immune responses in our FLA-definedsemi-inbred cats. This pilot MAP study showed only moderate IFNγresponses and T-cell proliferation to individual peptide after the 2ndboost (Table 18). No adverse effects were observed throughout the study.More importantly, after 10 CID₅₀ of FIV_(FC1) challenge, completeprotection in 1 of 4 cats and partial protection in 2 of 4 cats wereobserved in the 4× MAP1/MAP2 boosted Group 1 with the second lowestamount of Fp4-3 peptide. All non-vaccinated control (Group 4, n=4) and1× primed control (Group 3, n=3) groups were infected (Table 17).Partial protection consisted of a 3-6 wk delay in FIV detection and2-log lower viral set point compared to the control group. However, only1 of 2 cats with partial protection was observed in the 6×MAP1b/MAP1/MAP2 boosted Group 2b with the lowest amount of Fp4-3peptide. The 6× MAP1/MAP2 boosted counterpart Group 2a with the mostFp4-3 peptide had no protection. Since Group 2b was immunized initiallywith MAP1b instead of MAP1 and had one partially protected cat, thissuggested that the non-EC peptide Fp4-3 may be blocking protection andinstead enhancing infection.

To test the possibility that Fp4-3 peptide may be enhancing FIVinfection, we performed an in vitro FIV enhancing/inhibitory analysiswith all MAP peptides and immunogens. Most remarkably, significantenhancement of FIV infection was observed with peptide Fp4-3 (Fp4-3 vs.Positive Control, p<0.05), peptide FRT3-3 and MAP1, whereas peptideFRT3-4 (p<0.05) and MAP2 (p<0.01) significantly inhibited FIV infection(FIG. 35). Since peptide Fp14-1 and MAP1b had no significant in vitroeffect, peptide Fp4-3 in the MAP1 is most likely the cause of the invitro enhancement observed with MAP1. Another notable observation isthat the most significant inhibition was observed with MAP2 whichcorrelates with the strong inhibition observed with FRT3-4 (nodifference between FRT3-4 vs MAP2, p=0.184). Since MAP2 consisted ofpeptides FRT3-3 and FRT3-4 with a natural overlap (FIG. 34), suchoverlap may have blocked the enhancing activity of peptide FRT3-3 whileallowing the inhibitory activity of peptide FRT3-4. Hence, MAP2 was themost protective immunogen followed by MAP1b. Moreover, none of the MAPvaccinated cats had early detection or enhancement in viral loadcompared to the control cats. Perhaps, the strong immunity generated bythe MAP2 has blocked the enhancing activity of the MAP1 when boostedtogether (Group 1, Table 3). To test this possibility, in vitro study iscurrently undergoing to evaluate whether MAP2 can block FIV enhancingactivity of MAP1. Furthermore, more cats will be vaccinated with MAP1band MAP2 to determine the levels of cytotoxins generated to peptidesFp14-1, FRT3-3 and FRT3-4. Since these peptides have inducedCTL-associated cytotoxin expression in T cells from HIV⁺ subjects (FIGS.31A-31B; FIG. 5 in (Sanou et al. 2013)), these EC peptides may alsomediate their in vivo effects by inducing anti-FIV specific CTLactivity. The in vitro results together with in vivo efficacy resultsfurther suggest that our in vitro tests (enhancing/inhibitory assay;cytotoxin/cytokine analyses) can select epitopes with potent antiviralactivities which should decrease the number of protective EC epitopesneeded to be tested in in vivo studies and more efficiently produce aneffective T-cell based vaccine against FIV and HIV-1.

Evaluating the Direct Viral Enhancing or Inhibitory Effect of Fp9-3 andHp15 Peptides.

Based on our FIV enhancing/inhibitory (E/I) assay, we have determinednon-EC peptide Fp4-3 and EC peptide FRT3-3 significantly enhanced invitro FIV infection. However, only peptide Fp4-3 correlated with the invitro enhancement induced by MAP1. Since the in vitro E/I assay usesPBMC from naïve specific pathogen free (SPF) cats, it is unlikely thatthe in vitro stimulation of anti-FIV CTLs occurred in the culture andeliminated the infected cells. More likely, these enhancing orinhibitory peptides induced cytokines (enhancement: IFNγ, TNFα, GM-CSF;inhibition: IFNα, IL10), chemokines (inhibition: MIP1α, MIP1β, SDF-1,RANTES), or cellular restriction factors (inhibition: Trim5α, APOBEC3g)that either enhanced or inhibited FIV/HIV-1 infection. The twoidentified FIV-enhancing epitopes (Fp4-3 and FRT3-3 epitope peptides)(FIG. 35) also induced high levels of IFNγ production in the PBMC ofFIV-vaccinated cats (data not shown) but moderate levels to FRT3-3(Sanou et al. 2013) and modest levels to Fp4 pool (FIGS. 30A-30F) (Roffet al. 2015) in the PBMC of HIV⁺ subjects. IFNγ has been shown toenhance in vitro FIV and HIV-1 infections of naïve cat and human PBMCsrespectively (Tanabe and Yamamoto 2001; Yamamoto et al. 1986). IFNγ isproduced during inflammatory responses to HIV-1 infection (Ipp andZemlin 2013). Thus, high levels of antiviral cellular immune responsesare needed while minimizing both non-specific and viral-specificactivation of CD4⁺ T cells which can enhance viral replication (Lane2010). Removal of the enhancing epitopes on FIV and HIV-1 immunogenswill be critical to produce an effective HIV-1 vaccine. To accomplishthis goal, we will design a vaccine based on antiviral epitopes directedat inducing viral-specific CD4⁺ CTLs, CD8⁺ CTLs and polyfunctional Tcells, and identifying viral enhancing epitopes to remove from vaccinecandidates.

More importantly, our most recent in vitro studies demonstrated that ECepitope FRT3-4 directly inhibited the FIV infection. This observation iscompletely opposite of the FIV enhancement observed with EC epitopeFRT3-3. Notably the enhancing epitope overlaps with the inhibitoryepitope. Hence, careful epitope mapping must be performed to remove anyenhancing epitopes from FIV vaccine immunogen. Similar to FIG. 36A,repeated in vitro E/I studies demonstrated that peptides Fp14-3 andFp14-4 do not directly enhance or inhibit FIV infection. Therefore theseEC T-cell peptides especially the Fp14-4 peptide, which induced strongCD8⁺ T-cell proliferation in FIV-vaccinated cats (FIGS. 33A-33C), shouldbe next tested for its ability to induce FIV-specific cytotoxinexpression before in vivo challenge efficacy study. Its HIV-1counterpart peptide Hp15-3 induced high levels and frequency ofcytotoxin responses in T cells from HIV⁺ subjects (FIGS. 31A-31B) (Roffet al. 2015). Similarly, Hp15-1, the HIV counterpart of Fp14-1, inducedrobust cytotoxin levels and responder frequencies in the CD8⁺ T cellsfrom HIV⁺ subjects. Moreover, Hp15-1 strongly inhibited in vitro HIV-1infection, whereas Hp15-3 had no direct effect on HIV-1 infection.Consequently, EC peptide Hp15-1 followed by peptide Hp15-3 should beincluded as the HIV-1 vaccine immunogens.

Another remarkable observation is that FIV p24 epitope peptide Fp9-3inhibited in vitro infection of both FIV (FIG. 36A) and HIV-1 (FIG. 36B)but FRT3-4 inhibited only FIV. However these in vitro results indicatethat FRT3-4 does not enhance FIV and HIV-1 infections and may be apromising T-cell epitope for vaccine since it induced CTL-associatedcytotoxin expression in T cells from HIV⁺ subjects (Sanou et al. 2013).If it also induces FIV-specific CTL activity in PBMC from FIV-vaccinatedcats then this peptide will have direct and indirect modes of anti-FIVactivities. In conclusion, we now have EC peptides Fp14-1, FRT3-4, andFRT3-3/3-4 as protective T-cell epitopes against FIV, and EC peptidesHp15-1 and Hp15-3 as protective T-cell epitopes against HIV-1, whereascross-reactive Fp9-3 peptide may be useful against both FIV and HIV-1.We have also determined that palmitated MAP (Pam-MAP) is an excellentvehicle to deliver the peptide-based vaccine.

TABLE 15 Sequence conservation in EC p24 epitopes of Hp15/Fp14 pools A.Subtype-B HIV-1 Hp15 Peptides SEQ ID NO. IFN 8P 4P 8C 4C E/I NC Hp15-1359 RAEQASQEVKNWMTE + + + + + ↓ − Hp15-2 360     ASQEVKNWMTETLLV + −− + + n − Hp15-3 361         VKNWMTETLLVQNAN + + + + + ↓ + B.Subtypes/Strains SEQ ID NO. Fp14 Pool Counterparts Compared to UCD1HIV-1 UCD1 (B) 457 RAEQASQEVKNWMTETLLVQNAN Identity (Similarity)*****:****.************ HIV-1 (A) 481 RAEQATQEVKGWMTETLLVQNAN 91% (100%)*********************** HIV-1 (B) 457 RAEQASQEVKNWMTETLLVQNAN 100%(100%) *****:*:******:******** HIV-1 (C) 482 RAEQATQDVKNWMTDTLLVQNAN 87%(100%) *******:*************** HIV-1 (D) 483 RAEQASQDVKNWMTETLLVQNAN 96%(100%) **********:***:******** SIV CPZ 484 RAEQASQEVKTWMTDTLLVQNAN 91%(100%) ****... ******.***:**** SIV 485 RAEQTDAAVKNWMTQTLLIQNAN 74% (96%)Mac251/Mac239 ** :  ** ::.::* : *** FIV (A, B, D) 486DQEQNTAEVKLYLKQSLSIANAN 39% (70%) [Fp14] ** :  **:::.::* : *** FIV (C)487 DQEQNTAEVKTYLKQSLSLANAN 39% (74%) C. Subtype-B FIV Fp14 Peptides SEQID NO. IFN 8P 4P 8C 4C E/I NC Fp14-1 301 DQEQNTAEVKLYLK + + + + + − −Fp14-2 302   EQNTAEVKLYLKQSL − + − + + n − Fp14-3 303      AEVKLYLKQSLSIA + + + + + − − Fp14-4 488         KLYLKQSLSIANAN + + + + + − − Sections A and C show positive (+)or negative (−) response for IFNγ (IFN); CD8⁺ T-cell proliferation (8P);CD4⁺ T-cell proliferation (4P); CD8⁺ T-cell cytotoxins (8C); CD4⁺ T-cellcytotoxins (4C); or HIV enhancing or inhibitory (E/I) in HIV⁺ subjectswhen compared to those in HIV⁻ control group (NC). Symbols: inhibitory(↓); identical aa (*); closely (:) or moderately (.) similar aa; notdone (n); and bold aa residue differs from the one on HIV-1/UCD1.

TABLE 16 Sequence conservation in EC epitope FRT3-3 Subtypes/Strains SEQID NO. FRT3-3 Counterparts Compared to UCD1 HIV-1 UCD1 (B) 435KKKDSTKWRKLVDFRE Identity (Similarity) **************** HIV-1 (A) 435KKKDSTKWRKLVDFRE 100% (100%) **************** HIV-1 (B) 435KKKDSTKWRKLVDFRE 100% (100%) **************** HIV-1 (C) 435KKKDSTKWRKLVDFRE 100% (100%) **************** HIV-1 (D) 435KKKDSTKWRKLVDFRE 100% (100%) **************** SIV CPZ 435KKKDSTKWRKLVDFRE 100% (100%) ****..***.*:**** SIV MM 489KKKDKNKWRMLIDFRE 75% (100%) *** *:***.*:**** FIV (A, C) 490KKK-SGKWRMLIDFRE 75% (94%) *** *:***.*:*** FIV (B, D) [FRT3-3] 63KKK-SGKWRMLIDFRV 67% (87%)Strong IFNγ and T-cell proliferation responses to FRT3-3 decreased tozero when the following aa's on the FRT3-3 peptide were changed with thecorresponding ones on the HRT3-3 peptide: V15→E15, I11→V11, and M9→K9(highest to lowest decrease) (Sanou et al. 2013). The underline shows anepitope which is also detected by HIV⁺ subjects.

TABLE 17 Prototype Dual-Subtype FIV (IWV) Vaccine Prime and MAP VaccineBoosts Group # Full-to-Partial [# of cats ] Prime Boost-1 Boost-2Boost-3 Boost-4 Boost-5 Boost-6 Protection 1 [4] 1× IWV → MAP1 → MAP1 +2 → MAP2 → MAP1 + 2 — — ¾ (1 Full + 2 Partial) 2a [2] 1× IWV → MAP1 →MAP1 → MAP1 → MAP2 → MAP2 → MAP1 + 2 0/2 2b [2] 1× IWV → MAP1b → MAP1b →MAP1b → MAP2 → MAP2 → MAP1 + 2 ½ (Partial) 3 [3] 1× IWV — — — — — — 0/34 [4] PBS PBS PBS PBS PBS — — 0/4

TABLE 18 Prototype Dual-Subtype FIV (IWV) Vaccine Prime and Post-SECONDMAP Boost IFNγ (SFU) IL2 (SFU) Proliferation (CFSE^(low)) Cat ID PrimeBoost-1 Boost-2 Fp4-3/Fp14-l Fp4-3/Fp14-1 CD4⁺ T cells CD8⁺ T cells QVW1× IWV → MAP1 → MAP1 21/0 [0]  0/0 [9] 0/6 [7] 0/5 [6] DVD 1× IWV → MAP1→ MAP1 132/37 [206]  95/53 [296] 3/0 [2] 6/0 [9] QVQ 1× IWV → MAP1b →MAP1b  0/0 [43]  0/12 [165] 1/1 [7] 0/6 [7] DVB 1× IWV → MAP1b → MAP1b0/0 [0] 0/70 [76] 0/2 [1] 0/12 [10] Peptide Fp4-3 (left), peptide Fp14-1(middle) and MAP1 [right] were culture stimulants. IL2 and IFNγ ELISpotresults of >50 spot forming units (SFU/10⁶ PBMC) and CFSE-proliferationof >2 are considered positive (bolded). The prime-boost vaccinationinduced IL2 responses in 3 of 4 cats and IFNγ response in one. Largerresponses were induced by MAP1 than individual peptides in culture.These results demonstrate that both peptides in the MAP are recognizedby the cats and MAP formulation may be more effective in deliveringpeptides into cells.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

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I claim:
 1. A method for inducing an immune response in a person oranimal against an immunodeficiency virus, comprising administering oneor more antigens and/or immunogens to the person or animal, wherein saidone or more antigens and/or immunogens comprise one or moreevolutionarily conserved epitopes, wherein said epitopes are conservedbetween two or more different immunodeficiency viruses.
 2. The methodaccording to claim 1, wherein said epitopes are conserved between HIVand FIV.
 3. The method according to claim 1, wherein said epitopes areconserved between HIV, SIV, and FIV.
 4. The method according to claim 1,wherein said epitope is a T-cell epitope.
 5. The method according toclaim 4, wherein said T cell epitope induces one or more T cellresponses.
 6. The method according to claim 5, wherein said T cellresponse is release of cytotoxins and/or cytokines.
 7. The methodaccording to claim 1, wherein said epitope comprises the amino acidsequence of any of SEQ ID NOs:1-40 or any of SEQ ID NOs:45-591.
 8. Themethod according to claim 1, wherein said epitope comprises the aminoacid sequence of any of SEQ ID NOs:10, 21, 22, 23, 61, 62, 63, 64, 65,163, 164, 165, 166, 167, 176, 177, 178, 179, 214, 215, 216, 217, 218,288, 301, 303, 304, 359, 361, 431, 432, 438, 442, 443, 453, 459, 460,466, 479, 488, 492, or
 493. 9. The method according to claim 1, whereinsaid antigens and/or immunogens are peptides or proteins, and whereintwo or more peptides or proteins comprising the amino acid sequence ofany of SEQ ID NOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163, 164, 165,166, 167, 176, 177, 178, 179, 214, 215, 216, 217, 218, 288, 301, 303,304, 359, 361, 431, 432, 438, 442, 443, 453, 459, 460, 466, 479, 488,492, or 493 are administered to the person or animal.
 10. The methodaccording to claim 1, wherein said antigens and/or immunogens arepeptides or proteins, and wherein said peptides or proteins comprise twoor more amino acid sequences of any of SEQ ID NOs:10, 21, 22, 23, 61,62, 63, 64, 65, 163, 164, 165, 166, 167, 176, 177, 178, 179, 214, 215,216, 217, 218, 288, 301, 303, 304, 359, 361, 431, 432, 438, 442, 443,453, 459, 460, 466, 479, 488, 492, and/or
 493. 11. The method accordingto claim 1, wherein said epitope comprises the amino acid sequence ofany of SEQ ID NOs:10, 21, 22, 23, 176, 177, 178, 179, 214, 215, 216,217, or
 218. 12. The method according to claim 1, wherein said inducedimmune response is a CTL-associated immune response.
 13. The methodaccording to claim 1, wherein said induced immune response comprises aCD4+ and/or CD8+ T cell response.
 14. The method according to claim 1,wherein a person is administered said one or more antigens and/orimmunogens that are from an HIV and/or FIV.
 15. The method according toclaim 1, wherein the animal is a feline animal and is administered saidone or more antigens and/or immunogens that are from an FIV and/or HIV.16. A chimeric polypeptide comprising sequences of more than oneimmunodeficiency virus polypeptide.
 17. The chimeric polypeptideaccording to claim 16, wherein said polypeptide comprises matrix andnucleocapsid sequences of FIV and core or capsid sequence of HIV. 18.The chimeric polypeptide according to claim 16, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:43 or SEQ ID NO:44.
 19. Apolynucleotide that encodes a chimeric polypeptide of claim
 16. 20. Thechimeric polynucleotide according to claim 19, wherein saidpolynucleotide comprises the nucleotide sequence of SEQ ID NO:41 or SEQID NO:42.
 21. An antibody, or an antigen binding fragment thereof, that:i) binds specifically to an FIV protein or epitope and does not bind toan HIV protein or epitope; or ii) binds specifically to an HIV proteinor epitope and does not bind to an FIV protein or epitope; or iii) bindsto an FIV protein or epitope and also binds to the corresponding HIVprotein or epitope; or iv) binds to a peptide comprising an FIV or HIVepitope, wherein said epitope comprises an amino acid sequence of any ofSEQ ID NOs:1-40 or any of SEQ ID NOs:45-591.
 22. The antibody accordingto claim 21, wherein said antibody is a monoclonal antibody.
 23. Theantibody according to claim 21, wherein said antibody is the antibodydesignated as HL2309, HL2310, HL2350, or HL2351.
 24. A compositioncomprising: i) one or more epitopes evolutionarily conserved betweendifferent immunodeficiency viruses; and/or ii) one or morepolynucleotides that encode said one or more evolutionarily conservedepitopes; and/or iii) one or more chimeric polypeptides that comprisesequences from more than one immunodeficiency virus; and/or iv) one ormore chimeric polynucleotides that encode a chimeric polypeptide thatcomprises sequences from more than one immunodeficiency virus.
 25. Thecomposition according to claim 24, wherein said epitopes comprise anamino acid sequence of any of SEQ ID NOs:1-40 or any of SEQ IDNOs:45-591.
 26. The composition according to claim 24, wherein saidpolypeptides comprise the sequence shown in SEQ ID NO:43 or SEQ IDNO:44; or wherein said polynucleotide comprises the sequence shown inSEQ ID NO:41 or SEQ ID NO:42.
 27. The composition according to claim 24,wherein said epitope comprises the amino acid sequence of any of SEQ IDNOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163, 164, 165, 166, 167, 176,177, 178, 179, 214, 215, 216, 217, 218, 288, 301, 303, 304, 359, 361,431, 432, 438, 442, 443, 453, 459, 460, 466, 479, 488, 492, or
 493. 28.The composition according to claim 24, wherein said compositioncomprises two or more peptides, wherein said two or more peptidescomprise, independently, the amino acid sequence of any of SEQ IDNOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163, 164, 165, 166, 167, 176,177, 178, 179, 214, 215, 216, 217, 218, 288, 301, 303, 304, 359, 361,431, 432, 438, 442, 443, 453, 459, 460, 466, 479, 488, 492, or
 493. 29.The composition according to claim 24, wherein said compositioncomprises a peptide or protein that comprises two or more amino acidsequences of any of SEQ ID NOs:10, 21, 22, 23, 61, 62, 63, 64, 65, 163,164, 165, 166, 167, 176, 177, 178, 179, 214, 215, 216, 217, 218, 288,301, 303, 304, 359, 361, 431, 432, 438, 442, 443, 453, 459, 460, 466,479, 488, 492, and/or
 493. 30. The composition according to claim 24,wherein said epitope comprises the amino acid sequence of any of SEQ IDNOs:10, 21, 22, 23, 176, 177, 178, 179, 214, 215, 216, 217, or
 218. 31.The composition according to claim 24, wherein said composition furthercomprises a pharmaceutically acceptable carrier or diluent.
 32. Avaccine that comprises a composition of claim
 24. 33. A method fordetermining whether a feline animal has been vaccinated against FIV,said method comprising assaying a biological sample from a feline animalfor the presence of an antibody that binds specifically to an HIVantigen and does not bind to an FIV antigen, wherein the presence ofsaid antibody is indicative of vaccination against FIV infection.
 34. Amethod for selecting antigens and/or immunogens for use in a vaccineagainst an immunodeficiency virus, wherein the method comprisesselecting a target protein from two or more immunodeficiency viruses andidentifying evolutionarily conserved epitopes of the target protein,wherein the epitopes are identified by assaying a peptide from saidtarget protein of one of said viruses for induction of one or more Tcell responses, wherein one or more of the identified epitopes thatinduce one or more T cell responses are selected for use as an antigenor immunogen in the vaccine.
 35. The method according to claim 34,wherein said immunodeficiency virus is HIV or FIV.
 36. The methodaccording to claim 34, wherein said T cell responses are release of oneor more cytotoxins and/or cytokines.
 37. The method according to claim34, wherein said T cell response is a CTL response and/or a T helperresponse.